Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction and glycolytic switch in the rat lung

Pulmonary arterial hypertension is tightly associated with mitochondrial dysfunction [1, 2]. There are several different mechanisms of mitochondrial dysfunction that have been proposed to be involved in PAH pathogenesis, such as electron transport chain (ETC) dysfunction [3, 4], insufficient mitochondrial biogenesis [5, 6], altered mitochondrial membrane potential [7], increased mitochondrial reactive oxygen species (mROS) production [8, 9], mitochondrial DNA damage [10], and insufficient mitochondrial repair via fission/fusion or mitophagy [11‐13]. Most of those mechanisms are also interconnected in many ways. For example, an increased mitochondrial ROS and DNA damage could initiate the processes of fission/fusion, apoptosis or impaired biogenesis. The current work focuses specifically on ETC dysfunction based on its important roles in both ATP production by mitochondrial respiration and as a primary contributor to an increase in mitochondrial ROS. The resulted oxidative stress is known to be strongly incorporated into PAH pathogenesis due to its ability to induce DNA and protein damage. Finally, ETC dysfunction mediates an increase in the mitochondrial potential that has been previously described to mediate a glycolytic shift [14].

There are several clinical cases reporting that mitochondrial respiratory chain disease is accompanied by a manifestation of primary pulmonary hypertension (PPH) and severe pulmonary hypertension [15, 16]. The assessment of the respiratory complexes activity indicated a decreased Complex I and III activity in the tissues of these patients (liver tissue upon liver transplantation, skeletal muscle and skin fibroblasts obtained through biopsy). Thus, the inhibition of Complex III activity is a physiologically relevant stimulus to investigate whether ETC inhibition can induce pulmonary hypertension in a pre-clinical model. Mitochondrial respiratory complexes utilize iron-sulfur clusters to transfer electrons from donors to acceptors. It was also reported that the mutations in the iron-sulfur chaperon protein NFU1 have a strong association with pulmonary hypertension development [17]. All seven patients with NFU1 mutation developed fatal encephalopathy and pulmonary hypertension. Assessment of mitochondria respiratory complexes showed a decrease in Complexes I, II and III activities in patients with the NFU1 mutation, thus, pointing on respiratory chain failure as an important contributor in the development of pulmonary hypertension.

ETC can be affected by inhibition of any of its four known complexes. However, inhibition of Complex III was found to induce maximal ROS production and therefore maximal damage to mitochondria [18]. Electrons flow from Complex I or II to Complex III and then Complex IV generating a gradient of protons in the inner membrane. This gradient is utilized by ATP synthase to produce ATP or by uncoupling proteins to produce heat. Inhibition of Complex III can induce superoxide production in both the intermembrane space and the mitochondrial matrix [19]. Superoxide radical is usually scavenged by a mitochondrial form of superoxide dismutase - MnSOD and transformed into less active hydrogen peroxide (H₂O₂) [20]. However, nitric oxide (NO) produced in the vasculature can also scavenge superoxide to form peroxynitrite, a highly potent oxidant, which may inhibit ROS scavengers by induction of protein nitration and nitrosylation, like MnSOD [21], and initiate many pathophysiological pathways [22, 23].

Indeed, lungs of patients with PAH have possessed a ubiquitous nitrotyrosine staining [24, 25], which indicated a high level of peroxynitrite formation in PAH patients. Caveolin-deficient mice that are exhibited chronic pulmonary hypertension and severe right ventricle (RV) hypertrophy [26] had a high level of protein nitration in the lungs due to persistent endothelial nitric oxide synthase (eNOS) activation. Notably, the treatment of Cav1−/− mice with either an eNOS inhibitor or a superoxide scavenger reversed the PAH phenotype. In our recent work, we have reported that nitration mediates an over-activation of Akt with subsequent translocation of eNOS to the mitochondria. This, in turn, reduces the mitochondrial respiration and promotes endothelial dysfunction [27].

The primary female hormone estrogen is known to increase NO production due to an increase in both eNOS expression and activity [28, 29]. The vasodilatory, anti-inflammatory, and anti-thrombotic properties of NO provide the background for an advanced level of cardiovascular protection in females compared to males. At the same time, it could be expected that in the presence of mitochondria dysfunction and increased production of superoxide, the estrogen-mediated elevation of NO bioavailability can turn to be a damaging factor through an enhanced formation of peroxynitrite. Since the nitrosative stress has been linked with PAH development, it is possible that an enhanced protein nitration may explain the higher prevalence of PAH in females compared to males. Therefore, in this study, we utilized female rats to elucidate the consequences of repetitive inhibition of the ETC.

Under normal conditions, cells mainly rely on oxidative phosphorylation due to a highly efficient nature of mitochondria-mediated ATP production. However, the disease conditions associated with mitochondrial damage limits mitochondria’s ability to generate ATP and force the cells to switch to glycolysis [30, 31]. This metabolic shift towards a glycolytic metabolism, known as the Warburg effect, has been described in cancer cells, as well as highly proliferative pulmonary vascular cells [32, 33]. Importantly, in our previous research, we have confirmed that the metabolic transformation occurs even at the early stage of PAH when the physiological manifestation of the disease, such as RV pressure and hypertrophy, are still negligible [34]. Thus, the metabolic alterations significantly precede PAH manifestation and may be considered as a contributing factor for PAH development. The current work confirms that inhibition of the mitochondrial ETC is sufficient to initiate the same metabolic changes as seen in lungs of patients with PAH.

There is a vast body of literature from animal models to patients with PAH confirming that different types of mitochondrial dysfunction are strongly associated with PAH pathobiology [1, 2, 30, 31]. However, whether these alterations are directly triggered PAH or subsequent manifestations of the disease and contribute to PAH progression remains unclear. The current study demonstrates, for the first time, that the chronic inhibition of ETC is sufficient to initiate sustained pulmonary vasoconstriction, an increased vascular edema, and a glycolytic metabolic shift in healthy female rats. These observations strongly support the notion that impaired mitochondrial respiration can trigger PAH development.

Interestingly, when the effect of Crotalaria spectabilis plant seeds given orally was discovered to produce experimental PH in the 1960s, the early effects were described as acute pulmonary edema, infiltration of mononuclear cells and eosinophils, and pulmonary arteritis with edema of the pulmonary artery wall [38, 39]. Pulmonary edema at early stages of PAH (14 days after MCT i.p. injections) was also observed in another recent study, occurring at different anatomic levels (vascular edema, perivascular, bronchial, peribronchial, and alveolar septae) [40]. By day 28 after MCT injection, this edema was found to turn into vascular remodeling, suggesting that MCT induced damage of pulmonary arteries and subsequent edema may contribute to the initiation of uncontrolled vascular repair and remodeling at late stages. In this study, we found that chronic ETC inhibition also induces perivascular edema, which peaks at day 12 and mimics the histological changes seen in MCT and Sugen/hypoxia (our unpublished observation) models. We suggest that perivascular edema could contribute to vascular stiffness, impair the oxygen transport to the vascular wall and potentiate the activation of inflammatory cells. All these events (vascular stiffness, hypoxia, and inflammatory component) are known to be tightly associated with the increase in the pulmonary arterial pressure and contribute to the pathogenesis of PAH. Besides, this early edema may promote a late onset of vascular proliferation in PAH. Indeed, a subset of pulmonary arteries at days 12 and 24 has displayed the signs of remodeling. These observations support the idea that AA treatment is capable of inducing progressive pulmonary vasoconstriction even 18 days after the last AA application. The development of early lung edema may reflect either a direct deleterious effect of AA on pulmonary endothelial cells or initiated by an enhanced mitochondrial ROS production, which is known to induce p38 MAPK mediated endothelial barrier disruption [41]. Following the histological developments in these rodent models for a longer period of time is likely required to confirm this observation.

In this study, the chronic AA treatment did not result in the development of RV remodeling. While the occasional severe PAH without a detectable RV hypertrophy has been previously described for humans [42] and reported in a mouse model with PAH phenotype [43], the absence of RV hypertrophy did not allow us to consider the AA-induced condition as a developed pulmonary hypertension. Instead, we classify the AA-induced changes as an early stage of the disease mainly characterized by chronic pulmonary vasoconstriction. Nevertheless, the absence of RV hypertrophy may be attributed to the inhibitory effect of ETC dysfunction on myocardial contractility. In particular, the reduced levels of ATP production upon ETC inhibition, for example, can diminish RV reserve without an ability to respond to elevated afterload with hypertrophic remodeling. At the same time, the progressively increased RV afterload in combination with reduced RV contractility may be the primary reason for an elevated end-diastolic volume, potentially contributing to an elevated RVDP (Fig. 1c).

The high levels of NO bioavailability in females were expected to induce acute nitrosative stress via an increase in mitochondrial superoxide production induced by ETC inhibition. However, our protein nitration data were not revealing elevations at 30 min after AA injection. In contrast, there was a progressive accumulation of nitrated plasma proteins that correlated with a gradual disease progression. These data suggest that basal NO production may be insufficient to induce an increase in plasma protein nitration, although it may still produce higher levels of protein nitration in specific cell compartments. However, the progressive chronic vasoconstriction induced by AA treatment may be responsible for the further stimulation of NO production, which, on the background of superoxide leak, could initiate sustained nitrosative stress and may contribute to PAH progression. This notion was supported by a strong positive correlation between the total protein nitration in the plasma samples of female PAH patients and PVR, and a negative correlation with CO, which both are important markers of PAH severity (Fig. 4). We conclude that the increased level of NO bioavailability associated with female gender promotes accumulation of nitrated proteins specifically in female patients. Nevertheless, whether the observed nitrosative stress contributes to PAH progression or just could serve as a gender-specific marker of PAH progression remains to be elucidated.

PAH development and progression are characterized by changes in small circulation hemodynamics, lung and right heart morphology, altered signaling, transcriptomics, proteomics, and metabolism. We have recently reported that before an alteration in macro-parameters such as morphology and hemodynamics, we have observed metabolic changes, which predisposed organs to the pathological transition [34]. The glycolytic switch of metabolism in pulmonary vasculature is a well-accepted and characterized phenomenon in PAH. It was shown with a PET scanning of PAH patients that increased glycolysis in lungs and RV, similar to glycolytic changes seen in the tumors of cancer patients, correlates with the presence and severity of PAH [44, 45]. However, there is still debate whether the glycolytic switch is secondary to electron transport chain dysfunction or is upregulated due to pathologic normoxic HIF1a signaling activation. Our data indicate that ETC inhibition can turn on the glycolytic shift in the lungs. Indeed, AA-induced mitochondria dysfunction led to a robust upregulation of glucose uptake in the lung and resulted in an accumulation of several glycolytic pathway intermediates. Besides, we found that although based on ribulose-5-phosphate data, the PPP pathway was reduced in our animals; the ribose level was upregulated and can be fed into the glycolytic pathway via ribose conversion by ribokinase. It is important to note that glycolysis is elevated 12 days after induction of mitochondrial dysfunction but reduced at the late stage (24 days). Although primary glycolytic pathway is reduced at day 24, the lactate levels are still elevated. This can be explained by an activation or overexpression of ribokinase that can convert ribose via increased 3PG into production of lactate. Thus, AA-induced mitochondrial dysfunction can lead to the similar metabolic changes observed in other pre-clinical models and PAH patients.

Aerobic glycolysis is not a very efficient way for ATP generation when compared to the amount produced by mitochondrial respiration. However, the rate of glucose metabolism via glycolysis is higher than the complete oxidation of glucose in the mitochondrial respiratory chain [46]. Moreover, glycolysis induces a carbon flux toward the production of intermediates that are needed for biosynthesis of lipids, amino acids, porphyrins, and nucleotides leading to uncontrolled proliferation [47]. An upregulation of glycolysis is known to induce anabolic reprogramming of cells, leading to a higher rate of cell proliferation. Thus, the upregulation of glycolysis in response to mitochondrial dysfunction may become a key factor in vascular remodeling that would maintain the increased pulmonary resistance and pulmonary pressure [48]. It is important to emphasize that glucose uptake by lungs of AA-treated rats was found to strongly correlate with a pulmonary pressure, confirming the importance of glycolysis in PAH progression.

In conclusion, we believe that this study provides a novel and important insight into the pathogenesis of PAH. The results of our study suggest that the initial mitochondrial dysfunction is involved in the early changes associated with PAH, such as pulmonary vasoconstriction, vascular edema, and metabolic shift. Although these early pathological events have been previously described for other models of PAH, they have never been observed in response to direct mitochondrial inhibition. While further research is required to fully understand the role of mitochondria in the complex mechanisms of PAH initiation and progression, our study provides the novel evidence of the causal role of mitochondrial dysfunction in the initiation of pathological events associated with PAH.