Changes in mitochondrial dynamics are correlated with decreased mitochondrial respiratory enzyme activity and ATP abundance in hypoxic PH. Moreover, PASMCs from PA present dysmorphic mitochondria with reduced respiratory chain coupling, inefficient use of oxygen, and increased glycolysis [56]. These pathological changes were significantly attenuated in knockdown HIF-1α cells. The HIF-1α knockdown also significantly increases the mitochondrial length/width ratio, reducing the number of mitochondria, and the mitochondrial area [56].

After hypoxia stimulation, lung tissues and PASMCs present reduced mitochondrial respiratory complex IV and ATP, increased ROS production, and down-regulated mtDNA content, indicating hypoxia-induced deficiencies in energy metabolism and enzymatic activity of the respiratory complex [56]. Hypoxia also promotes mitochondrial retention in the perinuclear area, partial microtubule dissociation, anomalous fusion activity, and suppression of general fusion activity, leading to mitochondrial shortening. A key trigger for this response is the suppression of mitochondrial ATP production and the depletion of cellular ATP [74].

The expression of the fusion proteins MFN1/2 is significantly reduced in pulmonary artery tissues from rats with PH. In addition, MFN1/2 mRNA levels are significantly decreased in PASMCs after exposure to hypoxia compared to normoxia. These results suggest that mitochondrial fusion proteins MFN1/2 are involved in pulmonary vascular remodeling and PH [56]. The expression levels of the DRP1 fission protein also show a significant increase in PASMCs following hypoxia, both in vivo and in vitro. This suggests that the mitochondrial fission protein DRP1 is involved in pulmonary vascular remodeling. HIF-1α mediates the hypoxic activation of DRP1 to result in the induction of the mitochondrial pathway of proliferation and apoptosis in PASMCs via modulation of protein cell nuclear antigen (PCNA) and caspase-3 expression [56].

Nox4 is a major subunit of NADPH oxidase. Its expression is increased in murine models of hypoxia-induced PH in the pulmonary vasculature of patients with PH [75]. Hypoxia increases Nox4, which promotes the increase of mitochondrial hydrogen peroxide, promoting PH [76]. Nox4 silencing by siRNA causes reduction of ROS levels under normoxic and hypoxic conditions and suppresses the hypoxia-induced significant ROS increase in pulmonary adventitial artery fibroblasts [77].

Studies have also shown that Nox4 is found within mitochondria [78,79]. Nox4 modulates the activity of enzyme complexes within the electron transport chain (ETC) [80], in addition to its interaction with complex I, inhibiting its activity [9]. As hydrogen peroxide (H₂O₂) is the main product of Nox4 activity, the presence of active Nox4 in mitochondria can be expected to increase mitochondrial H₂O₂ levels. Koziel et al., demonstrated a significant decrease in the concentration of H₂O₂ in the mitochondria of Nox4-knockout cells [81]. As another subunit of NADPH oxidase, an important finding regarding Nox4 is its role in mitochondrial morphology, as in Nox4-knockout cells mitochondria revealed a characteristic of highly separated, non-interconnected networks [81]. Furthermore, the increase in mitochondrial Nox4 expression induces vascular SMC-mediated structural remodeling of the vascular wall due to the increase in ROS [82].

PKCε is a cytosolic protein that can be translocated to mitochondria in certain situations, even though little is known about this mechanism [83]. PKC-ε also appears to play an important role in mitochondrial morphology. An interesting study established that PKC-ε activation in renal proximal tubular cells induces mitochondrial dysfunction and fragmentation, energy deficit, ROS generation and cell death [84]. Rieske iron-sulfur protein (RISP) is a primary key factor in the generation of ROS originated from mitochondria. Our studies and others reveal that RISP is an important primary molecule for initiating hypoxia-induced [ROS]_mito generation in PASMCs, cardiac myocytes, and neuronal cells [85]. We further demonstrate that [ROS]_mito can subsequently activate cytosolic PKCε and then cell membrane NOX to induce further ROS generation; this ROS-induced ROS production ultimately cause massive increases in [ROS]_i in PASMCs [46,86].

RISP-mediated increase of intracellular ROS may subsequently inhibit voltage-gated potassium (Kv) channels, and activates transient receptor potential (TRP) channels, ryanodine receptors (RyRs) (especially RyR2) as well as inosol 1,4,5-trisphosphate receptors (IP3Rs) to evoke a large increase in [Ca²⁺]_i, leading to numerous cellular responses. It is worth pointing out that RyRs may mediate the hypoxic inhibition of KV channels, activation of TRP channels, and amplify IP3R-dependent Ca²⁺ release [87,88]. Our very recent investigations discover that RyR2-mediated Ca²⁺ release from the sarcoplasmic reticulum can further promote mitochondrial ROS generation [46].

The pathophysiologic relevance and therapeutic implications of defective mitochondrial fusion and excessive fission in PH have been widely studied. PASMCs play a critical and central role in the development, progression, and advancement of PH. The mitochondria in PASMCs play a critical role as vascular sensors of oxygen and thus may respond to a slight increment or decrement of oxygen tension in the pulmonary artery by activating hypoxic pulmonary vasoconstriction (HPV) [89,90]. HPV is the mechanism employed by the lungs to rectify any ventilation-perfusion mismatch [64]. A decline in oxygen levels in the pulmonary vasculature gives rise to vasoconstriction and shunts the blood-flow towards promoting better perfusion in well-ventilated lung areas. HPV is also important in optimizing partial pressure of CO₂ in certain lung conditions including atelectasis and pneumonia [91]. The PASMCs inside these arterial segments have mitochondria that behave as though they have been exposed to continuous hypoxia, and the pathophysiology of PH is centered in these same resistant arteries. In particular, the mitochondria in PH PASMCs exhibit poor metabolism because of transcriptionally mediated inhibition of mitochondrial pyruvate dehydrogenase and are fragmented as a result of an imbalance between mitochondrial fission and fusion.

The hypothesis known as the “redox hypothesis” proposes that the ROS production from the electron transport chain (ETC) complexes I and III in the mitochondria is altered in direct correlation to the level of alveolar PO₂, thereby initiating HPV in the human body [92]. The disruption of electron flow and reduction in levels of the diffusible second messenger H₂O₂ serve as markers of acute hypoxia [93]. The consensus among experts is that the mitochondria act as a primary oxygen sensor in the body, monitoring the levels of ROS in the system as an indicator of hypoxia. However, some dissenting opinions suggest that there may be a paradoxical increase in ROS levels during hypoxia [94].

A set of scientific data reveal that the synthesis of diffusible redox mediators, including radicals and peroxides, is diminished specifically in the resistance PASMC (as opposed to the conduit artery PASMC) during physiologic hypoxia (as opposed to anoxia). As such, hypoxia may lead to a decrease in the generation of ROS, which, in turn, causes inhibition of Kv channels, membrane depolarization, activation of voltage-gated L-type calcium (Cav1) channels, depolarizing the PASMCs. Vasoconstriction begins because of the calcium entry [64,92,95].

The occurrence of HPV in resistance PAs can be attributed to the distinctive capacity of PASMC mitochondria to regulate their ROS production [96]. This phenomenon is not observed in other arteries such as the renal arteries, where the production of ROS in response to changes in PO₂ is not significantly altered, and hypoxia results in arterial dilation instead [96]. The precise role of mitochondrial dynamics in HPV is yet to be fully comprehended. However, mitochondrial fission is an obligatory preliminary step in the mechanisms that precede changes in mitochondrial ETC function and ROS signaling in the ductus arteriosus [97]. Although HPV can cause acute PH and contribute to diseases like high altitude pulmonary edema in genetically predisposed people, it is imperative to understand that the underlying mechanism that leads to PH is ROS generation [98]. Hypoxia suppresses the mitochondrial pathways of ROS generation that underlie the oxygen-sensing function of mitochondria. More specifically, chronic hypoxia activates HIF-1α which in turn decreases mitochondrial H₂O₂ production and minimizes PO₂-sensitive ROS generation. This in turn decreases HPV [99].

PH related to chronic hypoxia may also primarily result from the remodeling of pulmonary vessels (ex. medial hypertrophy of small pulmonary arteries (<200 μm) in addition to HPV [64]. This vasculopathy in PH is caused by abnormalities in redox signaling (activated HIF-1 and decreased SOD2), oxidative metabolism (increased PDK and inhibited PDH), mitochondrial dynamics (increased dynamin related protein 1, DRP1, and reduced MFN2), and effector targets (altered expression of O₂-sensing K_v channels). Warburg hypothesis proposes that this impaired O₂-sensing impairment contributes to the underlying pathology in PH in a similar pattern like cancer relies on glycolysis despite availability of oxygen availability for oxidative metabolism. This theory suggests that both PH and cancer are reliant on the failure of oxygen-sensing due to the alteration of mitochondrial redox functions. This, in turn, manifests as further impairment of oxygen-sensing which in the longer term gives rise to the Warburg phenomenon [100].

The role of the Cyclin B and CDK1 complex has been investigated to play a role in PH. Cyclin B-dependent CDK1 initiates a cycle of mitosis by phosphorylating DRP1 at serine 616, therefore activating mitochondrial fission [101]. On the other hand, inhibition of mitotic fission arrests the cell-cycle at G2/M transition, promoting cell death [102]. However, whether hypoxic fragmentation of mitochondria leads to vasoconstriction in PH is still largely unknown. In ductus arteriosus, a change in PO₂ results in rapid (<60 s) mitochondrial fission, ultimately resulting in ROS production, inflammation, and vasoconstriction [97].

Von-Hippel Lindau disease (VHL), a genetic disease discovered in the Chuvash region of Russia, is a striking example of the impairment of oxygen-sensing in mitochondria as the underlying pathophysiology of pulmonary hypertension. A loss-of-function mutation in the VHL factor gives rise to pulmonary hypertension due to normoxic activation of HIF-1α [103,104]. The spectrum of symptoms in VHL comprises polycythemia and PH despite normal oxygen levels. This phenomenon demonstrates that impaired oxygen sensing (and the resulting normoxic activation of HIF-1α and HIF-2α) is sufficient to cause PH [103].

Mitochondrial redox signaling mechanisms in PH create a transcriptional and proteomic fingerprint like that observed in sustained hypoxia. These abnormalities are seen redundantly despite high-oxygen conditions, such as in cell-culture, which provide a state of pseudohypoxia.

PH-associated abnormalities in the pulmonary vascular oxygen-sensing pathway include persistent activation of HIF-1α during normoxic conditions [57] and a transcriptional activation of enzyme PDK in pulmonary arteries [57,96] and RV [105,106]. This causes a rapid shift from oxidative metabolism to aerobic glycolysis, and in turn, impaired mitochondrial fusion, and impaired fission, which results in the fragmentation of the PASMC’s mitochondrial network [102,107].