Hyperoxia-enhanced intermittent hypoxia conditioning: mechanisms and potential benefits

For nearly a century, repeated exposures to reduced levels of oxygen in the inspired air, such as exposure to high-altitude sojourns or breathing low O₂ gas mixtures, have been applied to induce adaptive mechanisms eliciting health benefits in humans. Intermittent hypoxia training, now more commonly referred to as intermittent hypoxia conditioning (IHC), was introduced in the 1930s to augment high-altitude acclimatization.1 Over recent decades, IHC has been increasingly reported to improve not only athletic performance, but also a variety of clinical conditions including cardiovascular and pulmonary diseases as well as metabolic and neurological disorders.1,2,3 IHC may be applied at rest (passive IHC) or combined with exercise (active IHC). Passive IHC is typically applied as repeated short (e.g., 3–10 minutes) exposures to moderately hypoxic gas mixtures (e.g., 14–10% O₂), interspersed with short (e.g., 3–10 minutes) intervals of normoxic (21% O₂) breathing.4

Understanding of the underlying mechanisms of IHC has been greatly expanded by the discovery and characterization of the hypoxia-inducible factors,5 for which William G. Kaelin Jr., Sir Peter J. Ratcliffe, and Gregg L. Semenza were awarded the 2019 Nobel Prize in Physiology or Medicine. In response to cellular hypoxia, these key transcription factors regulate the expression of an extensive portfolio of genes to orchestrate adaptive molecular processes thereby improving O₂ supply, reducing O₂ dependence, protecting cells from hypoxic/ischemic injury and increasing cellular resilience (Figure 1). Several reviews have summarized our growing yet still incomplete knowledge of the physiological and molecular mechanisms activated by hypoxia-inducible factors and other – often interdependent – mediators that produce the benefits of IHC but also potentially detrimental effects.3,6,7 In addition to hypoxia-inducible factors, the transcription factor nuclear factor erythroid 2-related factor 2 has been implicated in the health-promoting effects of hypoxia preconditioning by upregulating antioxidant mechanisms that prevent oxidative damage (Figure 1).8 Reactive O₂ species (ROS) generated during the abrupt hypoxia-to-normoxia transition have emerged as major factors that induce adaptations to IHC.8 Moderate increases in ROS trigger redox signaling cascades that mobilize the nuclear factor erythroid 2-related factor 2 gene program to effect adaptations that increase cellular, tissue and organism resilience.9 On the other hand, higher ROS levels impose oxidative stress that damages biomolecules and cells.

Figure 1.

Molecular signaling elicited by IHC and IHHC activates adaptive gene expression to confer health benefits.

Note: Moderate hypoxia activates the powerful transcription factors hypoxiainducible factor (HIF) and nuclear factor erythroid 2-related factor 2 (Nrf2) and their respective gene programs. Decreased cytosolic oxygen (O₂) concentrations stabilize HIF, allowing its translocation to the nucleus where it activates transcription of genes encoding an array of proteins, e.g., glycolytic enzymes, angiogenic factors and erythropoietin, that adapt the organism to hypoxic conditions. Also, hypoxiareoxygenation cycles cause the formation of reactive O₂ species (ROS). Upon reoxygenation, endoplasmic reticular nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase becomes the principal ROS generator. ROS liberate Nrf2 from its cytosolic binding complex, allowing its nuclear translocation and activation of a host of genes encoding antioxidant and anti-inflammatory enzymes and factors. Collectively, the products of the HIF and Nrf2 gene programs enhance metabolic control of serum glucose and lipids, increase physical exercise capacity, strengthen cellular ischemic resistance, improve memory function of cognitively impaired individuals, and increase cardiopulmonary function in patients with chronic cardiovascular and pulmonary diseases. Whether IHHC protocols induce such beneficial effects more efficiently than IHC requires confirmation. Created with Microsoft PowerPoint (version 16.0). IHC: Intermittent hypoxia conditioning; IHHC: intermittent hypoxia-hyperoxia conditioning.

Formation of ROS during hypoxia-normoxia transitions is rather modest, limiting the induction of protective antioxidant defenses. 10 Compared with normoxia, reoxygenation with moderate hyperoxia (intermittent hypoxia-hyperoxia conditioning, IHHC) may affect greater ROS production, including by nicotinamide adenine dinucleotide phosphate hydrogen oxidase in endoplasmic reticular membranes (Figure 1), thereby eliciting more robust induction of antioxidant genes.8,11 In patients with metabolic syndrome, IHHC proved to be a safe and readily tolerated intervention supporting the therapy and secondary prevention of components of metabolic syndrome and bolstering the patient’s anti-inflammatory status.12 As those studies did not directly compare IHHC with IHC, it remains unclear whether IHHC really confers benefits superior to IHC, and, if so, whether IHHC’s superiority may be due to more intense ROS formation and induction of antioxidant gene expression. Since ROS formation parallels increased O₂ concentrations, the therapeutic range of hyperoxia intensity likely has an upper limit, beyond which the cytotoxicity of excess ROS would outweigh the benefits. Our aim here is to summarize and evaluate the limited empirical evidence on this topic and propose future research to define potential safety and efficacy differences between IHC and IHHC.

Pre-clinical and clinical evidence on the superiority of IHHC compared to IHC: A recent meta-analysis reported safety and potential efficacy of both IHHC and IHC in healthy people and patients suffering from various diseases.13 When applied independently, both strategies increased exercise performance and tolerance in healthy and diseased subjects, particularly geriatric patients, and also effected improvements in patients suffering from cardiovascular and respiratory diseases, metabolic syndrome and/or cognitive impairments.13 Unfortunately, few studies have compared IHHC and IHC effects directly, especially in humans. One placebo-controlled trial evaluated the effects of 3-week IHHC and IHC programs in 55 prediabetic 51–74-year-old adults.14 Sessions consisting of 4 cycles alternating 5 minutes of hypoxia (12% O₂) and either 3 minutes of hyperoxia (IHHC, 33% O₂) or 5 minutes of normoxia (IHC) were administered 5 times a week.14 Compared to a sham protocol of continuous normoxia, IHHC and IHC produced similar reductions of serum glucose concentrations, total serum cholesterol and low-density lipoprotein levels.

Susta et al.15 evaluated short-term effects of IHHC and IHC on oxidative stress and antioxidant responses in 21 young (ages 1824 years), healthy men. In a crossover study design, participants were exposed to single IHHC and IHC sessions, 1 week apart, of 4–6 cycles of 5–7 minutes of hypoxia (11% O₂) followed by 3–5 minutes of 30–35% or 21% O₂, respectively.15 Neither single IHC nor IHHC sessions increased serum measures of oxidative stress or antioxidant capacity vs. the respective pre-treatment values,15 suggesting that multiple sessions of IHC or IHHC are necessary to accrue their full benefits.

While IHHC did not prove superior to IHC in these clinical studies, preclinical research in rodents has revealed different outcomes of IHHC vs. IHC. In rats, a 2-week program of daily, moderate IHHC (5 cycles alternating 5 minutes of 10% FiO₂ and 5 minutes of 30% FiO₂) generated more pronounced ROS signaling than an otherwise identical IHC protocol.16 IHHC pretreatment was associated with decreased basal and Fe²⁺/ascorbate-induced lipid peroxidation and formation of protein carbonyls and H₂O₂ in lung mitochondria during subsequent severe hypoxic stress (7% FiO₂ for 60 minutes). IHHC also induced gene expression and activities of Mn-superoxide dismutase and glutathione peroxidase more robustly than IHC, likely augmenting antioxidant protection. Other studies in rats compared the effects of eight swimming sessions alone or combined with IHHC or IHC.17,18 Normoxic swimming exercise improved exercise tolerance, increased rates of ROS detoxification and antioxidant enzyme activities in the heart, liver and brain. The combined IHHC-swim training attenuated lipid peroxidation in brain and myocardium during acute stress (swimming to exhaustion). Over-activation of heat shock proteins and the antioxidant enzymes superoxide dismutase and catalase were also prevented, relative to swim training alone or combined with IHC. The authors concluded that exercise combined with IHHC induces adaptations that limit oxidative damage of heart and brain more efficiently than exercise alone or exercise combined with IHC.

The collective evidence indicates that, although studies in rodents support the hypothesis that IHHC may induce beneficial adaptations more efficiently than IHC, a similar IHHC superiority in humans is not yet established. Only one study (and one protocol) directly compared the impacts of IHC and IHHC programs in humans, albeit on a limited set of metabolic variables.18 Susta et al.15 only applied single sessions of hypoxia and either normoxia or hyperoxia, so their results cannot be extrapolated to the effects of multi-session IHHC or IHC programs. Placebo-controlled studies comparing physiological responses and clinical outcomes of various IHHC and IHC protocols, and deciphering the signaling pathways initiated by IHHC vs. IHC, are urgently needed.

Outlook: clinical application of IHC vs. IHHC: IHC and IHHC are increasingly used to improve physical and, more recently, cognitive performance, and are potentially potent, essentially noninvasive treatments for various diseases. While several reports suggest that either IHC or IHHC may strongly benefit patients with therapeutically challenging, complex conditions, including mild cognitive impairment2 and hypertension,6 there are no clear recommendations for appropriate protocols. The lack of a harmonized terminology for beneficial applications of hypoxia19 and the complexity of the factors influencing the efficacy of intermittent hypoxia strategies are impeding the clinical applications of IHC or IHHC. Differences in the hypoxic dose (i.e., duration, intensity, number and frequency of hypoxia exposures), the heterogeneity of individual responses to hypoxia (which also raisese the question of whether fixed inspiratory O₂ levels or target arterial O₂ saturation values20 are preferable for research and clinical applications), as well as unclear effects of potential modulating factors (e.g., wakefulness/sleep and related circadian effects, physical activity and fitness, blood CO₂ levels, medications, age and sex) complicate optimization of IHC and IHHC protocols. Here we focused on one specific parameter, the use of normoxic vs. hyperoxic intervals between hypoxic exposures. Although data in rats suggest that IHHC might be the superior protocol, consistent with the presumed greater induction of ROS-mediated cellular responses leading to stronger adaptations, there is currently no evidence of this superiority in humans.16,18

In human studies, direct comparisons of IHC- and IHHC- induced physiological responses (e.g., cardiovascular, ventilatory, metabolic, neurophysiological) and cellular effects (e.g., oxidative stress, inflammation, energy metabolism, mitochondrial functions) will be crucial to substantiate IHHC’s potential superiority over IHC. Moreover, evidence-based recommendations on the hypoxic and hyperoxic doses need to be established for different populations. Typically, 30–35% O₂ is applied during the hyperoxic periods, but this moderate hyperoxia may be too weak a stressor to trigger meaningful responses to the hyperoxia. On the other hand, more intense hyperoxia may elicit excessive, potentially harmful ROS formation. The possibility that hyperoxia might interfere with aspects of hypoxia responses, e.g., by reversing hypoxia-inducible factor activation, also merits consideration, as does the likelihood that IHC or IHHC protocols eliciting maximally beneficial responses and adaptations may vary among individuals depending on age, sex, genetic background, physical fitness, health history, medications and other variables. The persistence of the health benefits following completion of IHC or IHHC programs is another pivotal factor. Consequently, more systematic evaluation of potentially health-promoting IHC/IHHC protocols is mandatory to maximize the benefits of these promising interventions for many diseases for which effective treatments are currently limited or unavailable.