As one of the important organelles for protein production and secretion in vivo, changes in endoplasmic reticulum homeostasis can lead to intracellular unfolded protein responses, resulting in mitochondrial protein imbalance. Studies have shown that mitochondrial protein imbalances can lead to metabolic disease, inflammation, neurodegenerative diseases and cancer (Wang and Kaufman, 2012). However, the relationship between mitochondrial protein imbalance and PAH is currently unclear. But James and others have suggested that mitochondrial protein homeostasis may play an important role in the pulmonary circulation (James et al., 2020). In this study, the authors found that altered mitochondrial proteostasis promotes glycolysis and initiates metabolic reprogramming by reducing protein clearance and detoxification, resulting in sustained constriction and proliferation of pulmonary vasculature (James et al., 2020).

During mitochondrial biogenesis, mitochondrial DNA (mtDNA) is synthesized under the control of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). In this process, the mitochondrial transcription factor a (TFAM) nuclear gene is the final effector of mtDNA transcription and replication. PGC-1α can increase the expression of TFAM by stimulating nuclear respiratory factor-1 (NRF-1), nuclear respiratory factor-2 (NRF-2) and estrogen-related receptor-α (ERR-α) (Jornayvaz and Shulman, 2010; Popov, 2020). Hypoxia, loss of peroxisome proliferator-activated receptor-γ (PPARγ), or decreased PGC1α reduced mitochondrial mass by increasing mitochondrial hydrogen peroxide production and mitochondrial fission, thereby impairing mitochondrial biological function and promoting PASMCs proliferation (Yeligar et al., 2018).

As a nicotinamide adenine dinucleotide-dependent deacetylase, Sirtuin 1 (SIRT1) can enhance mitochondrial biogenesis by deacetylating and activating PGC-1α. One study found that SIRT1 activators inhibited the proliferation of mouse and human PASMCs under hypoxia-induced conditions, and SIRT1 knockout mice exhibited higher vascular remodeling than non-knockout mice. This effect is related to the activation of mitochondria caused by the overexpression of mitochondrial markers and the down-regulation of PGC-1a (Zurlo et al., 2018). Additionally, the vasodilator nitric oxide (NO) controls cellular respiration and mitochondrial biogenesis (Afolayan et al., 2016). In vascular smooth muscle cells, NO is an effective vasodilator. It promotes PASMCs and vasodilation by activating soluble guanylate cyclase (sGC) to increase the production of cGMP, thereby reducing calcium influx (Furchgott and Zawadzki, 1980). In a fetal sheep model of persistent PAH, inhaled NO can reduce mitochondrial DNA copy number, electron transport chain complex subunit and adenosine triphosphate levels to promote mitochondrial biogenesis, and improve mitochondrial dysfunction (Afolayan et al., 2016).

Nitric oxide synthase isoenzymes include neuronal NOS, inducible NOS and endothelial NOS (eNOS) (Coggins and Bloch, 2007). Among them, eNOS mainly comes from pulmonary vascular endothelium and is also the main source of NO in pulmonary circulation. Many studies suggest that the production of NO in PAH is significantly reduced. Although many studies have found that the production of NO in PAH patients is impaired, their results are contradictory (Coggins and Bloch, 2007; Lázár et al., 2020). The reason is that some studies found that chronic hypoxia can reduce the expression of eNOS in pulmonary vessels (Ziesche et al., 1996; Ostergaard et al., 2007), while other studies found that chronic hypoxia can induce the expression of all NOS subtypes, including eNOS (Fagan et al., 2001; Ward et al., 2005). Lázár et al. (2020) study have shown that the decreased expression or dysfunction of endothelial nitric oxide synthase (eNOS) can eliminate the NO signal transduction in PAH. It can be seen that targeted inhibition of eNOS degradation may repair mitochondrial function, thereby reducing the occurrence and development of PAH. Another study also has shown that miR-1226-3p can target Profilin-1 to increase the content of eNOS and NO in PAH rats, thereby protecting rats from PAH induced injury (Jian and Xia, 2021). It can be seen that the role of eNOS in PAH is controversial at present, and more research is needed to confirm it.

The phenomenon of continuous division and fusion of mitochondria is called mitochondrial dynamics. Even under metabolic and environmental pressures, mitochondrial fusion can still transfer gene products between mitochondria to achieve optimal function. Mitochondrial division is crucial to maintain the number and proper distribution of mitochondria in daughter cells (Adebayo et al., 2021). When this balance is disturbed, it may cause many diseases, including cancer, PAH, etc.

Mitochondria promote mitochondrial fusion through optical atrophy 1 (OPA1) and mitofusin 1 (MFN1) and mitofusin 2 (MFN2) between the outer and inner membranes of adjacent mitochondria. MFN1 and MFN2 mediate mitochondrial outer membrane fusion, while OPA1 mediates mitochondrial inner membrane fusion (Sabouny and Shutt, 2020). Inhibition of MFN1, MFN2 or OPA1 will reduce mitochondrial fusion and promote mitochondrial fission. Therefore, MFN1, MFN2 or OPA1 may also be targets for intervention in PAH.

Increased MFN2 expression can increase mitochondrial fusion, reduce mitochondrial fission, reduce PASMCs proliferation, and increase PASMCs apoptosis (Lu et al., 2016; Zhu et al., 2017). Ryan’ study (Ryan et al., 2013) found that decreased mitochondrial fusion is also a complementary means of mitochondrial division, and the study identified MFN2 down regulation as a proliferation pathway. In human PAH and two established rodent PAH models, MFN2 deficiency led to excessive proliferation of PASMCs. The basis of MFN2 downregulation is related to the down-regulation of PGC1a, a transcriptional coactivator of MFN2. Reduced expression of MFN2 and PGC1a contributes to mitochondrial.

MFN1 is involved in the proliferation of PASMCs in patients with PAH (Omura et al., 2019). Ma et al. (2017) showed that hypoxia upregulated the expression of MFN1 in PASMCs both in vivo and in vitro, and that MFN1-mediated mitochondrial homeostasis and PASMCs proliferation were regulated by miR-125α. This also provides a theoretical basis for the treatment of PAH through miR-125α-MFN1 pathway. Recently, researchers found that O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic amidoxim (BGP-15) promotes mitochondrial fusion by promoting the GTPase activity and self-aggregation of OPA1, but this role has not been confirmed in PAH (Szabo et al., 2018).

Mitochondrial health is primarily maintained through mitochondrial dynamics and mitophagy. Mitophagy plays an important role in the quality control of mitochondria by eliminating damaged mitochondria. Fission can also isolate the damaged and irreparable mitochondria and promote mitophagy. Therefore, mitochondrial dynamics and mitophagy are closely related. Mitophagy is a form of selective autophagy. When mitochondrial damage occurs, the translocation mediated by TIM23 complex is inhibited, resulting in the retention of the transmembrane domain of PTEN-induced putative kinase 1(PINK1), its integration into the outer membrane, escape from degradation, autophosphorylation, and phosphorylation. This leads to the accumulation and activation of E3 ubiquitin-protein ligase parkin (Parkin) (Hoshino et al., 2019), which promotes the ubiquitination of mitochondrial outer membrane proteins (Ordureau et al., 2014). This ubiquitination of mitochondrial outer membrane proteins leads to the participation of autophagy receptors to activate mitophagy. Autophagy receptors include CALCOCO2 (also known as NDP52), Optineurin (OPTN), Tax1-binding protein (TAX1BP1), and FUNDC1 (Lazarou et al., 2015; Liu et al., 2022). FUNDC1 belongs to the mitophagy receptor. Study Liu et al. (2022) found that the overexpression of FUNDC1 can promote the mitochondrial phagocytosis and cell proliferation of PASMCs by activating ROS-HIFA pathway, thus leading to pulmonary vascular remodeling. Study Ma et al. (2022) found that apoptosis-inducing factor (AIF) deficiency in hypoxia can lead to mitochondrial complex I instability, resulting in oxidative phosphorylation (OXPHOS) disorder, high levels of mitochondrial ROS production, and increased expression of Pink and Parkin, leading to excessive mitophagy.

However, some studies have found that UCP2 is also involved in mitochondrial autophagy. As a negative ion transport protein, uncoupling protein 2 (UCP2) is mainly located in the inner membrane of mitochondria. Loss of UCP2 will lead to the reduction of Ca²⁺ from the endoplasmic reticulum into mitochondria in PASMCs, leading to the reduction of mitochondrial Ca²⁺, and then inhibit mitochondrial function. A prospective study found that UCP2 was positively correlated with the severity of PAH. Furthermore, the mean pulmonary artery pressure and vascular remodeling in Sirt3 and Ucp2 double knockout PAH mice were significantly increased, and the mitochondrial function in pulmonary arteries was significantly inhibited. UCP2 of pulmonary vascular endothelial cells also can induce mitochondrial phagocytosis, insufficient mitochondrial biosynthesis and increased endothelial cell apoptosis through PTEN and kinase 1, thus leading to pulmonary vascular remodeling (Haslip et al., 2015).

It is well known that under hypoxia, the function of mitochondrial ion channels in PASMCs will be significantly affected, resulting in impairment of various functions including mitochondrial oxidative phosphorylation and ATP synthesis efficiency, regulation of signal transduction and enzyme activity in cells and cell matrix, as well as cell proliferation and apoptosis (Wang X. et al., 2021). Mitochondria are composed of the inner and outer mitochondrial membranes and the mitochondrial matrix and membrane gap (Mukherjee et al., 2021). Among them, the mitochondrial inner membrane has high ion selectivity, and its permeability plays an important role in maintaining the ion concentration inside and outside the mitochondria. The selectivity of the outer membrane of the mitochondria is low, allowing free ingress and egress of ions (Singh, 2021). The abnormal ion concentration gradient on the inner and outer membrane of mitochondria is significantly related to the occurrence and development of early pulmonary artery contraction, late pulmonary artery remodeling, myocardial ischemia-reperfusion injury and other diseases (Wang X. et al., 2021).

Mitochondrial ion channels include calcium (Ca²⁺), potassium (K⁺), sodium (Na⁺) and some anion channels. Anion channels can participate in the occurrence and development of cardiovascular diseases by maintaining the energy and material metabolism balance of mitochondria and cells, and regulating the calcium cycle of mitochondria. Mitochondrial K⁺ channels have two biological functions. One is to regulate the K⁺ concentration in mitochondria to maintain the stability of mitochondrial volume, promote the expression of enzymes related to mitochondrial energy metabolism, and protect cells. On the other hand, it participates in the oxidation and phosphorylation of mitochondria (Moudgil et al., 2006). The uptake of K⁺ through this channel can partially compensate for the charge change caused by the proton pump transport of H⁺, thus maintaining the stability of mitochondrial transmembrane potential. K⁺ channels may also participate in pulmonary vascular tone and remodeling. Despite all this, Calcium channel is the most important channel. Calcium channels are the most accurate and mature (Alevriadou et al., 2017).

The contraction of PASMCs caused by hypoxia is the key factor to trigger hypoxic pulmonary vasoconstriction, and the increase of intracellular Ca²⁺ level is one of the main reasons for the contraction of PASMCs (Jain et al., 2020). Hypoxia can induce the release of Ca²⁺ from intracellular calcium stores, promote the uptake of Ca²⁺ by mitochondria, and lead to the increase of the concentration of Ca²⁺ in cells, thus triggering hypoxic pulmonary vasoconstriction. Mitochondria in PASMCs mainly act as sensors in physiological hypoxia (Zhao Enqi and Ye 2021).

Ca²⁺ uptake by mitochondrial must pass through the outer and inner membrane of mitochondria. Hydrogen ions move through the mitochondrial inner membrane through the electron transport chain to form a concentration gradient. It accumulates electrochemical driving force for calcium ion transport to mitochondria. Mitochondrial Ca²⁺ uniporter (MCU) is the main pathway of mitochondrial Ca²⁺ uptake, which exists in the inner membrane of mitochondria. Mitochondrial Ca²⁺ uptake-1 (MICU1) is a part of MCU complex, and only Ca²⁺ is allowed to enter mitochondria. Ca²⁺ first passes through the outer membrane of mitochondria through voltage dependent anion channel 1, and then passes through the inner membrane of mitochondria through MCU to enter mitochondria (Klapper-Goldstein et al., 2020). MCU is highly selective to Ca²⁺, which is the main way for mitochondria to absorb Ca²⁺ (Baradaran et al., 2018). When the concentration of Ca²⁺ in the cytoplasm is low, MCU1 can prevent mitochondria from taking Ca²⁺ by setting a threshold, thus playing the role of gatekeeper (Payne et al., 2017).

Studies have shown that mitochondrial membrane potential, as a driving force, promotes the transfer of Ca²⁺ to mitochondria (Woods et al., 2019). MCU mediated mitochondrial uptake of Ca²⁺ plays an important role in signal transduction, bioenergetics, and cell contraction, proliferation, and apoptosis. Its imbalance is related to a variety of human diseases. Hypoxia can lead to the release of Ca²⁺ from intracellular calcium stores, and lead to the influx of extracellular Ca²⁺ by stimulating calcium channels on the cell membrane, thus leading to intracellular Ca²⁺ overload. MCU can remove excessive Ca²⁺ in the cytoplasm, and mitochondria also use these ions to generate cell energy (Baughman et al., 2011). Persistent hypoxia can damage the function of MCU, reduce the concentration of ROS in mitochondria, and then promote the contraction and proliferation of PASMCs.

Mitochondrial associated endoplasmic reticulum (MAMs) is the physical basis of communication between endoplasmic reticulum (ER) and mitochondria (Ahsan et al., 2021). The transfer of Ca²⁺ from ER to mitochondria depends on the specific calcium channels on the MAMs surface. The site where calcium stores release Ca²⁺ forms high calcium cytoplasm at MAMs and reaches a high concentration of Ca²⁺ in a short time, which is much higher than the concentration of Ca²⁺ in cytoplasm. Ca²⁺ in ER and sarcoplasmic reticulum (SR) can be released into mitochondrial matrix through inositol triphosphate receptors (IP3R) or ryanodine receptors (RyR) on their surfaces. Endoplasmic reticulum mitochondria interaction occurs in the whole network, and Ca²⁺ is one of the most important signals used by organelles for communication (Zhao Enqi and Ye 2021). The Ca²⁺ released by SR is the key to excitation contraction coupling (Sydykov et al., 2021).

The main site of ROS production induced by hypoxia is respiratory chain complex III. Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction (Rafikova et al., 2018). Hypoxia can induce the production of ROS in mitochondria and increase the concentration of Ca²⁺ in PASMCs, thereby promoting the uptake of Ca²⁺ by mitochondria in PASMCs, which may play an important role in hypoxic pulmonary vasoconstriction and its related PAH (Yang et al., 2020). Studies have shown that the Riske Fe/S protein in complex III can mediate the production of ROS during hypoxia, thereby increasing the level of Ca²⁺ in PASMCs (Truong et al., 2019). RU360, an inhibitor of mitochondrial Ca²⁺ uptake, inhibits mitochondrial Ca²⁺ uptake by inhibiting the production of ROS induced by hypoxia in mitochondria, and finally inhibits hypoxia induced mitochondrial Ca²⁺ overload. Under hypoxia, Riske Fe/S protein dependent mitochondrial ROS production in respiratory chain complex III can activate phospholipase C-γ 1 causes IP3 generation, IP3R channel opens and releases Ca²⁺. In addition, the levels of Endothelin-1 (ET-1) and ET-A in PAH patients were increased. ET-A stimulates calcium release into the cytoplasm by increasing the concentration of IP3, leading to PASMCs contraction. ET-B, on the other hand, reduces the concentration of IP3 to cause vasodilation and clearance of ET-1 (Chaumais et al., 2015). Therefore [ROS] mito plays an important role in hypoxia induced Ca²⁺ release of SR and contraction of PASMCs. The increase of [Ca²⁺] mito will produce a large amount of ROS, which will lead to the opening of mitochondrial permeability transition pore, and finally lead to the increase of [Ca²⁺]i, forming a positive feedback mechanism. At the same time, ROS and redox couple in PASMCs can also inhibit oxygen sensitive voltage gated potassium channel Kv, activate voltage gated calcium channel, and then increase [Ca²⁺]i, leading to PASMCs contraction and hypoxic pulmonary vasoconstriction.

It has been shown that hypoxic stimulation of pulmonary vasoconstriction is due to inhibition of mitochondrial function as cellular oxygen receptors (Michelakis et al., 2004). The intracellular ROS include free radical superoxide (O₂-), hydroxyl radicals (OH-), non-free radical hydrogen peroxide (H₂O₂), and oxygen negative ions (O-), which are mainly generated from mitochondria, reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxygenase and nitric oxide synthase (Khan et al., 2021).

Mitochondrial respiratory chain is the main source of biological ROS (Skulachev, 2007). The active oxygen produced mainly refers to the single electron reduction products of oxygen, O₂- and its derived HO₂-, OH-, H₂O₂, and singlet oxygen (Mao Min et al., 2014). In addition, there are many enzyme systems in the organism that can convert O₂ into O₂-, such as the redox protein p66SHc between the inner and outer membranes of mitochondria, which uses the reduction amount of respiratory chain and oxidizes cytochrome C to produce H₂O₂, which is a new way to generate ROS in the mitochondrial respiratory chain (Giorgio et al., 2005).

Both mitochondrial cytochrome P450 and NADPH oxidase play an important role in regulating ROS production. Nox1-5 and Duox1-2 of NADPH family on the outer membrane of mitochondria are involved in the regulation of ROS production. In addition, cyclooxygenase, xanthine oxidase and lipid oxidase also affect ROS production in varying degrees (Mao Min et al., 2014). Many studies have found that many signal transduction pathways such as nuclear factors-κB (NF-κB), MAPK, phosphatidylinositol 3-kinase and p53 are all involved in ROS production. For example, tumor necrosis factor can activate apoptosis signal regulated kinase, increase ROS production, and up regulate the downstream influence factor c-Jun amino terminal kinase, thus activating the p38-MAPK pathway to induce cell contraction and apoptosis (Bigarella et al., 2014).

Mitochondria, as the primary consumption area of oxygen in cells, have always been regarded as oxygen sensing sites. They are released into the cytoplasm in the form of O₂- and H₂O₂ signals, triggering downstream transcriptional signaling factors, such as hypoxia inducible factors, NF-κB and signal transducer and activator of transcription, thereby inducing pulmonary vascular inflammation, endothelial and smooth muscle contraction, and vasoconstriction and remodeling (Frazziano et al., 2014).

The ubiquity of ROS determines the universality of its damage to body functions, and it can be used as a signal molecule to affect downstream ion channels, such as the following oxygen sensitive voltage gated potassium channels, which increase potassium ions in the cytoplasm, depolarize pulmonary artery smooth muscle cells, thus activating voltage gated Ca²⁺ channels, and Ca²⁺ influx leads to hypoxic pulmonary vasoconstriction (Trebak et al., 2010).

Hypoxic PAH is a common systemic inflammatory syndrome, including scleroderma, systemic lupus erythematosus and pulmonary vascular disease (Balabanian et al., 2002). ROS plays an important role in the pathological changes of vascular diseases and is an important agonist of inflammatory response (Irani, 2000; Lennon and Salgia, 2014). ROS may promote the contraction and proliferation of PASMCs by increasing the secretion of proinflammatory chaperone cyclosporine binding protein A (Satoh et al., 2008). The increase of ROS production can promote the adhesion and migration of dendritic cells, and pretreatment with antioxidants can significantly inhibit this pathological phenomenon (Zhu et al., 2009).

Autophagy is a cellular process that relies on lysosomes to remove damaged organelles, maintain the stability of the intracellular environment, and enhance the tolerance of cells to starvation or hypoxia. ROS can induce autophagy, among which H₂O₂ and O₂- are the most important inducers. The occurrence of mitochondrial autophagy can be reduced by reducing the content of O₂- in mitochondria, which may be related to the ion release and lipid peroxidation induced by aconitase (Bensaad et al., 2009; Chen et al., 2009).

When mitochondria are damaged, toxic substances can be gathered into one mitochondria and then autophagic degradation can be induced through division (Aggarwal et al., 2013). Mitochondrial respiratory chain complexes I and II can regulate autophagic cell apoptosis through mitochondrial ROS, which further confirms the close relationship between mitochondrial ROS and autophagy (Chen et al., 2007). In addition, autophagy damage can negatively feedback to increase oxygen pressure, promote the accumulation of ubiquitinated proteins, ROS production and mitochondrial damage.

In the PAH rat model, inhibition of mitochondrial fusion protein DRP1/NADPH oxidase (NOX) pathway and autophagy related protein (Atg) - 5/Atg-7/Declin-1/microtubule related protein light chain 3 (LC3) dependent autophagy pathway in PASMCs can reduce PASMCs proliferation (Wu et al., 2019). ROS scavenger N-acetylcysteine can inhibit the formation of autophagy, and autophagy inhibitor chloroquine or 3-MA can also reduce the production of ROS, indicating that autophagy and the production of ROS have a close regulatory and feedback regulatory relationship, but the specific mechanism needs further study (Teng et al., 2012).

Hypoxia can directly or indirectly promote the secretion of regulatory factors to regulate the blocking and activation of various kinase activities and signal pathways, thereby aggravating the contraction and remodeling of pulmonary arteries. Hypoxia can increase the calcium sensitivity of smooth muscle cells and regulate the intracellular Ca²⁺ concentration by activating Rho/Rho kinase signal, thereby promoting pulmonary vasoconstriction (Takemoto et al., 2002; Absi et al., 2019).

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is expressed in many tissues and organs in vivo, and its biological activity is closely related to a variety of cell functions, including proliferation and oxidative stress reaction (Matsumoto, 2019). It is found that activation of ALDH2 can reduce IL-1β, IL-6 and inflammatory protein one levels in serum of PAH mice, thereby reducing the process of PAH (Zhao et al., 2019). Calcineurin (CaN) is a serine/threonine protein phosphatase that depends on calcium and calmodulin. When the level of calcium ions in the cytoplasm increases, calcium ions can activate CaN by binding regulatory subunits and activating calmodulin binding. CaN activates the transcription factor T cell nuclear factor of activated T cells (NFATc) through dephosphorylation, and the activated dephosphorylated NFATc is then transported to the nucleus to further exert its function (Roy and Cyert, 2020). NFAT is a transcription factor family, including five members: NFATc1, NFATc2, NFATc3, NFATc4, and NFAT5. In recent years, some types of NFAT have been found to play an important role in the pathological process of hypoxic PAH. Jiang et al. (2018) found that NFATc3 was significantly increased in PASMCs under the hypoxia conditions. It has been found that mitochondrial ALDH2 and CaN can directly bind and regulate each other, and CaN also has a direct binding relationship with NFATc3 (Chen, 2021). Therefore, mitochondrial ALDH2 can inhibit the proliferation and contraction of pulmonary vascular smooth muscle cells by mediating the expression of CaN/NFATc3, thereby improving the contraction of pulmonary vessels in the acute phase and remodeling in the chronic phase (Chen, 2021).