Carbon dioxide (CO₂) is a metabolic product of cellular oxidative respiration, and is primarily eliminated from the blood and tissues by the lungs under physiological conditions. An elevation in CO₂ partial pressure in arterial blood over 45 mmHg is termed hypercapnia. Increased CO₂ levels are often observed in conditions where an impairment of the alveolar-capillary barrier function or a decline in alveolar ventilation occurs (Vadasz et al., 2012b; Herold et al., 2013). Various acute and chronic lung diseases, such as acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), asthma and cystic fibrosis are frequently accompanied by hypercapnia (Vadasz et al., 2012b; Radermacher et al., 2017). Furthermore, elevated CO₂ levels and intermittent hypoxia combined with hypercapnia play a role in the pathogenesis of obstructive sleep apnea, atherosclerosis and obesity (Kikuchi et al., 2017; Imamura et al., 2019; Xue et al., 2021).

It is increasingly evident, that various non-excitable cells, such as alveolar epithelial cells, fibroblasts and immune cells are sensitive to the changes in CO₂ concentrations independently of intra- and extracellular pH, reactive oxygen species (ROS) and involvement of the carbonic anhydrases (Putnam et al., 2004; Shigemura et al., 2017; Cummins et al., 2019). In contrast to earlier reports, which suggested that hypercapnia might be tolerated or even beneficial in the setting of critically ill patients (Fuller et al., 2017; Roberts et al., 2018); more recent studies have shown that elevated CO₂ levels are associated with higher complication rates, increased risk of exacerbations, more severe disease states, worse outcomes and an increased risk of mortality both for acute and chronic lung diseases (Yang et al., 2015; Nin et al., 2017; Husain-Syed et al., 2020; Shigemura et al., 2020). In addition, translational studies established that high CO₂ levels impair alveolar fluid clearance, innate immunity and cellular host defense, decrease cytokine production, downregulate phagocytosis and macrophage activity. Hypercapnia also stimulates nitric oxide (NO) production, therefore negatively impacting on pulmonary metabolism, aggravates epithelial cell repair, alters cellular lipid metabolism, decreases muscle anabolism, increases smooth muscle airway contractility and muscle catabolism, thus contributing to disease states and impaired recovery (Lang et al., 2000; Vadasz et al., 2008; Gates et al., 2013; Jaitovich et al., 2015; Kikuchi et al., 2017; Shigemura et al., 2018; Korponay et al., 2019). In addition, recent studies suggest that elevated CO₂ levels increase mortality in animal models of acute lung injury secondary to viral and bacterial insults (Gates et al., 2013; Casalino-Matsuda et al., 2020).

Protein transcription, translation, folding, and maturation continuously occur in each living cell and are essential for normal physiological function. In the cell, approximately one-third of the proteome and most of the secretory and membrane proteins are processed through the endoplasmic reticulum (ER) (Brodsky and Skach, 2011). In addition, the ER regulates lipid metabolism and serves as an intracellular calcium store (Hetz et al., 2015; Schwarz and Blower, 2016). The ER coordinates numerous co- and post-translational protein modifications, including N-linked glycosylation, formation of disulfide-bonds, sequence cleavage, chaperone-assisted protein folding, recognition and targeting of the ER-localized proteins for degradation (Ellgaard and Helenius, 2003; Araki and Nagata, 2011; Ellgaard et al., 2018). Numerous ER-resident chaperons, such as calnexin, calreticulin and binding immunoglobulin protein (BiP) orchestrate co-translational folding/refolding of nascent proteins. In addition, these chaperons play a central role in the removal of terminally misfolded proteins via ER-associated degradation (ERAD) and are key players of unfolded protein response (UPR) during ER stress (Hebert and Molinari, 2007; Halperin et al., 2014). Up to date, three main UPR pathways, named by ER-localized proteins have been characterized: inositol-requiring enzyme 1 (IRE1), protein kinase RNA-activated (PKR)-like ER kinase (PERK), and activating transcription factor-6 (ATF6). An increase of misfolded/unfolded proteins in the ER leads to dissociation of BiP from ER stress sensors, autophosphorylation of the sensors and subsequent activation of UPR (Wang and Kaufman, 2016; Almanza et al., 2019).

Of note, ER stress plays a pivotal role in the pathomechanism of various respiratory diseases, including but not limited to COPD (and in particular cigarette smoke exposure), viral and bacterial pneumonia, asthma, interstitial lung diseases and cystic fibrosis (Korfei et al., 2008; Lawson et al., 2011; Kenche et al., 2013; Kim et al., 2013; van ’t Wout et al., 2015; Lee et al., 2016; Marciniak, 2017; Tang et al., 2017; Schmoldt et al., 2019), many of which are accompanied by hypercapnia (Vadasz et al., 2012b; Shigemura et al., 2020). Notably, these disease states also often lead to hypoxia. Indeed, low oxygen levels have also been shown to negatively impact ER homeostasis, thus inducing ER stress (Chipurupalli et al., 2019; Bradley et al., 2021). Although the effects of hypoxia on the ER lie beyond the scope of the current manuscript, it is increasingly evident that hypoxia negatively affects ER function in alveolar epithelial cells and macrophages in the lung. These effects involve the downregulation of metabolic processes and disruption of the ER chaperone activity, which result in activation of key elements of the UPR, such as PERK, eIF2a, and IRE1α (Burman et al., 2018; Delbrel et al., 2018, 2019; Diaz-Bulnes et al., 2019; Bradley et al., 2021). Another cellular organelle that is tightly related to the ER is the peroxisome (Dimitrov et al., 2013). Of note, recent publications suggest that hypercapnia affects peroxisome signaling by modulation of the activity and expression of peroxisome proliferator-activated receptors (Huang et al., 2016; Kikuchi et al., 2017). At the molecular level, elevated CO₂ has been shown to activate kinases and proteins that are known to regulate ER function and/or participate in UPR, such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK1/2), AMP-activated protein kinase (AMPK), B-cell lymphoma 2 (Bcl-2), and caspase-7 (Vadasz et al., 2008, 2012a; Welch et al., 2010; Casalino-Matsuda et al., 2015; Dada et al., 2015; Shigemura et al., 2018). Furthermore, recent reports suggest that CO₂ can impact post-translational protein biochemistry by carbamate formation and subsequent protein carbamylation (Meigh et al., 2013; Linthwaite et al., 2018). In this review, we will focus on the molecular mechanisms by which hypercapnia impairs protein folding in the ER. Unfolding/misfolding of proteins in the ER by elevated CO₂ levels result in enhanced protein retention or degradation, thereby impairing subsequent protein trafficking, and thus overall cellular and tissue function.

It is well documented that protein maturation in the ER requires a specific milieu, including high Ca²⁺ levels, sufficient amounts of ATP, and an appropriate oxidizing environment (Jager et al., 2012; Almanza et al., 2019). In particular, in the past two decades, a number of studies revealed that disruption of the ER folding environment leads to accumulation of misfolded/unfolded proteins, induces ER stress and subsequent activation of the UPR (Araki and Nagata, 2011; Wang and Kaufman, 2016).

A consequence of protein misfolding/unfolding in the ER, is ER stress and subsequent activation of IRE1α-, PERK-, and ATF6-mediated UPR pathways. The UPR response may be adaptive or maladaptive, depending on the markedness and duration of the stimulus (Wang and Kaufman, 2016). The adaptive mechanisms “aim” to restore the protein folding homeostasis in the ER by downregulating protein synthesis, activating ERAD and modulating function of specific ER chaperones. If the initial UPR response does not allow coping with ER stress, the maladaptive arm of UPR will be activated that may lead to cellular death, mostly via apoptosis (Wang and Kaufman, 2016).

A numbers of studies have shown that physiological ER stressors selectively activate UPR branches, thereby triggering non-classical stress responses within the ER, which do not lead to cellular death and have rather adaptive character (Raina et al., 2014; Bergmann et al., 2018). In line with this notion, we now know that exposure of alveolar epithelial cells to hypercapnia transiently activates IRE1α and induces ERAD of the ER-resident β-subunit of the Na,K-ATPase, thereby decreasing plasma membrane abundance of the transporter (Kryvenko et al., 2021b). Furthermore, enhanced protein degradation in the ER by ERAD is associated with increased ubiquitination of the target protein, which has been shown to occur upon hypercapnia (Gwozdzinska et al., 2017). Of note, a recent study identified the IRE1α interacting partner, TNF receptor-associated factor 2 (TRAF2), as a novel E3-ligase involved in the polyubiquitination of the Na,K-ATPase β-subunit (Gabrielli et al., 2021). However, whether TRAF2 is additionally required for ERAD of the Na,K-ATPase will need to be addressed in future studies. Interestingly, treatment of cells with CO₂ levels of up to 120 mmHg for a duration of 5 days is not associated with increased apoptosis or cellular death in alveolar epithelial or mesenchymal cells (Vohwinkel et al., 2011), suggesting that at least in these settings of hypercapnia a rather adaptive type of UPR is activated.

It is well documented that elevated CO₂ levels initiate specific signaling cascades in cells, including activation of ERK1/2, JNK, and AMPK-α₁ that drive retrieval of the Na,K-ATPase and epithelial sodium channel (ENaC) from the plasma membrane, thereby causing alveolar epithelial barrier dysfunction and altering alveolar fluid balance (Vadasz et al., 2008; Welch et al., 2010; Gwozdzinska et al., 2017). Moreover, exposure of skeletal muscles to increased CO₂ concentrations leads to stimulation of AMPK-α₂ and is associated with a decrease in protein synthesis and increased muscles catabolism (Jaitovich et al., 2015; Ceco et al., 2017; Korponay et al., 2019). In general, AMPK activation is a response to metabolic stress by sensing AMP:ATP and ADP:ATP ratios, aiming to reestablish energy balance by reducing anabolic processes that require ATP and by promoting catabolic mechanisms that generate ATP (Garcia and Shaw, 2017). In contrast, in the setting of short-term hypercapnia, AMPK activation is independent of the metabolic status of the cell and is rather secondary to intracellular Ca²⁺ signaling (Vadasz et al., 2008). Notably, knockdown of AMPK in bronchial epithelial cells leads to a significant increase in CHOP levels resulting in ER stress and apoptosis (Liu et al., 2018). Moreover, AMPK activation downregulates BiP levels induced by tunicamycin or thapsigargin and has been found to regulate ER and mitochondrial morphology upon stress conditions, thus preventing mitochondrial fragmentation and apoptosis (Wikstrom et al., 2013; Kim et al., 2015).

Extracellular signal-regulated kinase, a member of the mitogen-activated protein kinase (MAPK) family, has been shown to play an essential role in UPR by interacting with IRE1α and by promoting transcription of pro-survival anti-apoptotic proteins, such as myeloid leukemia cell differentiation protein-1 (Mcl-1), Bcl-2 and B-cell lymphoma-extra large protein (Bcl-xL) (Darling and Cook, 2014). Furthermore, activation of ERK1/2 has been shown to be cytoprotective upon ER stress, by downregulating cellular apoptosis upon thapsigargin- and tunicamycin-induced UPR (Arai et al., 2004; Hu et al., 2004).

In addition, several other mechanisms may contribute to the adaptive or maladaptive signals upon hypercapnia. For example, hypercapnia has been found to inhibit autophagy in human macrophages by increasing expression of Bcl-2 and Bcl-xL, thus blocking Beclin-1 apoptotic complex formation (Casalino-Matsuda et al., 2015). Of note, Bcl-2 is involved in the regulation of ER calcium homeostasis und upregulation of the molecule may play a protective role upon ER stress by lowering steady-state levels of ER Ca²⁺ via InsP3R activation (Sano and Reed, 2013). However, the anti-apoptotic effects of Bcl-2 are inhibited by JNK (Sano and Reed, 2013) that is markedly upregulated in the setting of acute and chronic hypercapnia (Vadasz et al., 2012a; Dada et al., 2015; Gwozdzinska et al., 2017). In fact, the role of activated JNK, in contrast to AMPK and ERK1/2, is usually associated with an enhanced pro-apoptotic ER stress response. On the other hand, JNK is involved in the downstream cascade of IRE1α activation, the UPR branch responsible for preventing ER overload by ERAD (Maurel et al., 2014; Prischi et al., 2014; Almanza et al., 2019). Recently, hypercapnia has been associated with increased airway smooth muscle contractility in the setting of asthma, which is mediated by activation of caspase-7, an apoptosis-related cysteine peptidase (Shigemura et al., 2018). Interestingly, caspase-7 has also been found to be involved in the ER-stress mediated cell death upon thapsigargin treatment and caspase-7 ablation was able to reprogram the UPR and reduced JNK-induced apoptosis (Dahmer, 2005; Choudhury et al., 2013).

Thus, while activation of AMPK, ERK1/2, JNK, and caspase-7 drive clearly deleterious (maladaptive) signals leading to cellular dysfunction upon hypercapnia, activation of these signaling molecules may, at least in part, limit further injury by reducing the elevated CO₂-induced ER stress, as part of an adaptive mechanism.

Protein maturation and folding in the ER require a specific milieu, which depends on Ca²⁺, ATP and an oxidative environment. Recent studies focusing on the pathophysiological effects of hypercapnia established that elevated CO₂ levels alter the ER folding machinery. The molecular mechanisms driving ER dysfunction upon high CO₂ concentrations include reduced cellular ATP levels, a Ca²⁺ disbalance in the ER, as well as altered redox homeostasis of the organelle (Figure 3). These events lead to ER stress, UPR, and ERAD of target proteins, potentially resulting in tissue and organ malfunction. To what extent these signals are adaptive or maladaptive depend on the extent and duration of hypercapnia and require further experimental assessment.

VK and IV drafted, edited, and approved final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.