Under phase contrast microscopy, HACMs exposed to 72 hours of intermittent hypoxia or severe hypoxia appeared more flat and spindle shaped than cells incubated under normoxia or mild hypoxia for the same period of time (Fig. 2, A–D). Cultures exposed to intermittent hypoxia for 8 hours showed a reduced cell count to those exposed to mild hypoxia (P < 0.01; Fig. 3A), whereas cultures exposed to mild hypoxia exposed for 8 hours showed an increased cell count (P < 0.05). Furthermore, cultures exposed to intermittent hypoxia for 8 hours released more LDH than cells exposed to mild hypoxia (P < 0.05) or normoxia (P < 0.05), indicating a more rapid cytotoxic response (Fig. 3B). After 24 hours, the cell count was similar in cultures exposed to intermittent hypoxia, mild hypoxia, and normoxia but significantly lower in cultures exposed to severe hypoxia (P < 0.05). Similarly, more LDH was released from cultures exposed to severe hypoxia compared with normoxia or mild hypoxia after 24 hours (P < 0.05). After 72 hours, cell count was reduced in cultures exposed to intermittent hypoxia and severe hypoxia than in cultures exposed to mild hypoxia (P < 0.01) or normoxia (P < 0.0001). Furthermore, intermittent, mild, and severe hypoxia exposure resulted in greater LDH release compared with all other groups after 72 hours (P < 0.01). Thus, intermittent hypoxia resulted in a more rapid and severe cytotoxic response than sustained hypoxia of the same average O2 or normoxia.
VEGF release, as estimated by the accumulation in the medium, was higher in HACM cultures exposed to severe hypoxia for 24 hours compared with cultures exposed to normoxia (P < 0.0001; Fig. 3C). Compared with normoxia, extracellular VEGF accumulation was higher after 72 hours of exposure to mild hypoxia (2-fold, P < 0.05), intermittent hypoxia (9-fold, P < 0.0001), or severe hypoxia (12-fold, P < 0.0001). After 8 hours, IL-1β secretion was higher in cultures exposed to intermittent hypoxia compared with mild hypoxia (P < 0.05; Fig. 4A). IL-1β release after 24 hours was highest from cultures exposed to severe hypoxia compared with all other groups (P < 0.05). IL-1β release after 72 hours was greater from cultures exposed to intermittent hypoxia and to severe hypoxia than from those exposed to normoxia (P < 0.0001) or mild hypoxia (P < 0.0001). Similar patterns could be observed for IL-6 and IL-8 secretions, revealing significant increases after 72-hour exposure to intermittent hypoxia compared with mild hypoxia (P < 0.05) or normoxia (P < 0.0001; Fig. 4, B and C). Furthermore, cultures exposed to severe hypoxia showed greater MIF release after 24 hours than cells exposed to normoxia (P < 0.05) or mild hypoxia (P < 0.05). After 72 hours, MIF release was higher in cultures exposed to intermittent hypoxia and severe hypoxia compared with mild hypoxia (P < 0.05) or normoxia (P < 0.05).
Nineteen genes showed significant changes in mRNA abundance after 24 hours of hypoxia (Table 1). The expression levels of 4 genes in the hypoxia signaling pathway (lactate dehydrogenase A [LDHA], vascular endothelial growth factor A [VEGFA], carbonic anhydrase IX [CA9], WNT1 inducible signaling pathway protein 1 [WISP1]) were upregulated in cultures exposed to severe hypoxia. In contrast, WISP1 expression was downregulated in cultures exposed to intermittent hypoxia. The expression of 3 genes including the antiapoptotic member of the BCL2 gene family (BCL2L1), the gene encoding glutamate cysteine ligase (GCLC), and the gene encoding plasminogen activator inhibitor-1 (SERPINE1) were selectively downregulated by intermittent hypoxia. Furthermore, the expression levels of Dab, mitogen-responsive phosphoprotein homolog 2 (DAB2), growth arrest and DNA-damage-inducible beta (GADD45B), Hes family BHLH transcription factor 1 (HES1), interferon regulatory factor 1 (IRF1), v-myc avian myelocytomatosis viral oncogene homolog (MYC), wingless-type MMTV integration site family, member 5A (WNT5A) were downregulated in cultures exposed to intermittent hypoxia and severe hypoxia but not in cultures exposed to mild hypoxia.
We demonstrated that intermittent hypoxia (0%–21% O2) exerts deleterious effects on HACMs, as evidenced by lower cell count and higher LDH, VEGF, and cytokine (IL-1β, IL-6, IL-8, and MIF) secretion. Moreover, cell inflammation and injury occurred earliest in the intermittent hypoxia group. Severe hypoxia showed more detrimental effects on HACMs at later time points. Pathway analysis showed that intermittent hypoxia exposure activated the oxidative stress, Wnt, Notch, and hypoxia pathways. Our in vitro results suggest that intermittent hypoxia, occurring during OSA or other respiratory-related diseases, may have early injurious effects on human myocardial cells equal to or partially exceeding those induced by constant severe hypoxia.
Intermittent hypoxia may occur in a number of clinical scenarios, including the interruption of myocardial blood flow and disorders such as OSA, chronic obstructive lung disease, smoking, sickle cell anemia, or immaturity of newborns.3,4 In addition, cyclic recruitment and derecruitment of atelectasis during mechanical ventilation can result in intermittent hypoxia.12–18 This phenomenon occurs because of repetitive opening and closing of atelectasis (cyclic atelectasis), thereby causing varying pulmonary shunt fraction. In subjects with lung disease, cyclic atelectasis can cause within-breath arterial partial pressure of O2 oscillations that are transmitted with the circulation to organs, exposing them to intermittent hypoxia.19–21
The underlying mechanisms by which OSA-related intermittent hypoxia causes injury to the myocardium have been partially investigated. OSA has been described to be an independent risk factor for heart failure, arrhythmias, and myocardial infarction.3–5 In contrast, the effect of intermittent hypoxia caused by cyclic atelectasis on the myocardium presently remains unknown. Although cyclic atelectasis has not been demonstrated in clinical settings, some evidence hints toward its existence in patients undergoing high-risk surgery because arterial partial pressure of O2 levels can fluctuate, despite hemodynamic steady-state conditions.22,23 Importantly, intermittent hypoxia caused by either OSA or cyclic atelectasis may largely differ in frequency, amplitude, or range and therefore may exhibit different effects on the cardiovascular system and organs. As an example, brief periods of intermittent hypoxia can promote protection by hypoxic preconditioning, whereas long-term intermittent hypoxia such as during OSA can exhibit harmful cardiovascular and cerebrovascular effects.3,4,24 These associations of intermittent hypoxia are complex; therefore, research needs to identify the detailed characteristics of intermittent hypoxia in more detail.
Cell Count and Viability
Numerous studies have investigated the effects of constant hypoxia on murine cardiac myocytes. However, few studies have examined HACMs, and no previous investigation has compared the cytotoxicity of sustained versus intermittent hypoxia in HACMs. The present study reveals that exposure of HACMs to intermittent hypoxia resulted in more injury than exposure to constant hypoxia of the same average O2, irrespective of the duration. Moreover, intermittent hypoxia induced the greatest injury within the first 8 hours. Consistent with our findings, previous studies have reported chronic intermittent hypoxia-induced myocardial injury in rodents.25–27 However, other preclinical studies reported growth-promoting effects by intermittent hypoxia and protection against subsequent severe hypoxia.28–30 As highlighted earlier, discrepancies may result from differences in the severity, frequency, and/or duration of intermittent hypoxia exposure, and intermittent hypoxia can exert dichotomous (harm or benefit) effects on the human body.31
The secretion rates of IL-1β, IL-6, IL-8, and MIF, which are widely accepted mediators of myocardial inflammation and injury, were higher under intermittent and severe hypoxia than under mild hypoxia. This finding may account, in part, for enhanced cytotoxicity.3,4,32 Notably, an increase in IL-1β, promoting inflammation, occurred earliest in intermittent hypoxia-treated HACMs. Although the effects of IL-6 and IL-8 on cardiac myocytes remain poorly understood, it is widely accepted that IL-8 promotes chemotaxis, which could enhance the infiltration of other inflammatory cells into the myocardium.33,34 Alternatively, IL-6 reduces cardiac myocyte apoptosis, suggesting that its activation is an adaptive mechanism to promote survival under intermittent hypoxia.35 MIF has been shown to be expressed by myocardial cells in response to redox stress.36 Furthermore, an increase in MIF serum levels has been discussed as playing a role in the pathogenesis of myocardial ischemia.36,37
The expression levels of LDHA, CA9, and VEGFA, 3 well-described genes of the hypoxia pathway, were upregulated by mild and severe hypoxia but not by intermittent hypoxia.29 Furthermore, WISP1, a member of the CYR61/CTGF/Nov family of growth factors mediating cell proliferation, transformation, and survival, was upregulated by severe hypoxia but downregulated by intermittent hypoxia. Thus, cytoprotective factors induced by sustained hypoxia may not be induced by intermittent hypoxia. In fact, WISP1 has antiapoptotic effects on cardiac myocytes and regulates postinfarction heart remodeling.38,39 The BCL2 family members are key regulators of mitochondrial apoptosis.40 The BCL2 family members BAX and BCL2 were downregulated by severe hypoxia, whereas BCL2L1 was downregulated selectively by intermittent hypoxia, indicating that the activation of these apoptotic regulators is sensitive to the magnitude and kinetics of hypoxia. These differential gene expression patterns may also account for the observed differences in cell count and cytotoxicity among the treatment groups.
Furthermore, we measured the changes in the expression of genes related to oxidative stress signaling. The gene encoding thioredoxin reductase-1 was downregulated under all 3 hypoxic conditions, whereas the gene encoding GCLC was downregulated selectively by intermittent hypoxia. Glutamate cysteine ligase is a rate-limiting enzyme for the synthesis of glutathione, a crucial antioxidant in mammalian cells. Weakness in this antioxidant defense mechanism exposes cells to greater oxidative stress and increases the risk of myocardial infarction.41 Furthermore, we observed selective downregulation of SERPINE1, the gene encoding plasminogen activator inhibitor-1, after exposure to intermittent hypoxia, suggesting that intermittent hypoxia may also influence plasmatic coagulation.11
We acknowledge several limitations of our study. O2 was delivered via diffusion through RAMs, which limited the number of O2 oscillation cycles to 3 per hour that is slower than respiration but near the frequency of OSA. Classical cell culture models have investigated key mechanisms associated with intermittent hypoxia by O2 diffusion through the medium.29,42,43 This approach, however, likely requires longer to achieve full O2 equilibrium in the medium than O2 diffusion via RAMs. Therefore, higher intermittent hypoxia cycling frequencies cannot be easily achieved in in vitro models without changing the medium, which also has disadvantages such as mechanical stress on cells and dilution of the medium.44,45 Another important confounder is the O2 concentration used. To produce a measureable effect in vitro, we selected a range of O2 (0%–21%) causing a level of transient hypoxia. Previous investigations revealed that it is very difficult to reduce O2 solitarily by diffusion.46 Therefore, we doubt that real cellular anoxia was induced in the present model. This idea might be supported by our results because we measured 0% ± 0.5% O2 and not true anoxia in the severe hypoxia group. In addition, not all cells died in the severe hypoxia group even after 72 hours, indicating that cells still had enough O2 for survival. Nonetheless, our model provides a foundation for testing specific hypotheses regarding the differential impacts of continuous versus intermittent hypoxia on HACMs.
In HACMs, intermittent hypoxia (0%–21% O2) and constant severe hypoxia (0% O2) resulted in inflammation and injury, as revealed by reduced cell numbers and elevated LDH, VEGF, IL-1β, IL-6, IL-8, and MIF secretion. Cell inflammation and injury occurred earlier in HACMs exposed to intermittent hypoxia than in cultures exposed to the same average O2 (10% O2) or normoxia (21% O2). Severe hypoxia showed more detrimental effects on HACMs at later time points. Furthermore, intermittent hypoxia resulted in a distinct gene activation pattern that included members of the oxidative stress, Wnt, Notch, and hypoxia pathways. Our in vitro findings suggest that intermittent hypoxia exposure, as occurring during OSA or other respiratory-related diseases, may produce rapid and substantial damage to the human heart.
Name: Jing Wu, MD.
Contribution: This author helped conduct the study, analyze the data, and prepare the manuscript and also contributed equally to the work.
Attestation: Jing Wu approved the final manuscript.
Name: Joanna Stefaniak, MD.
Contribution: This author helped conduct the study, analyze the data, and prepare the manuscript and also contributed equally to the work.
Attestation: Joanna Stefaniak approved the final manuscript.
Name: Christina Hafner, MD.
Contribution: This author helped conduct the study, analyze the data, and prepare the manuscript.
Attestation: Christina Hafner approved the final manuscript.
Name: Johannes Peter Schramel, DI, DVM.
Contribution: This author helped design the study and equipment, prepare the manuscript, and in technical assistance.
Attestation: Johannes Peter Schramel approved the final manuscript.
Name: Christoph Kaun.
Contribution: This author helped prepare the HACMs cell culture and in technical assistance.
Attestation: Christoph Kaun approved the final manuscript.
Name: Johann Wojta, PhD.
Contribution: This author helped design and supervise the study prepare the manuscript.
Attestation: Johann Wojta approved the final manuscript.
Name: Roman Ullrich, MD.
Contribution: This author helped design and supervise the study prepare the manuscript.
Attestation: Roman Ullrich approved the final manuscript.
Name: Verena Eva Tretter, DI, PhD.
Contribution: This author helped design and conduct the study, analyze the data, and prepare the manuscript.
Attestation: Verena Eva Tretter approved the final manuscript.
Name: Klaus Markstaller, MD.
Contribution: This author helped design and supervise the study and prepare the manuscript.
Attestation: Klaus Markstaller approved the final manuscript.
Name: Klaus Ulrich Klein, MD.
Contribution: This author helped design and conduct the study, analyze the data, prepare the manuscript, supervise the study, fund the study, and is the archival author.
Attestation: Klaus Ulrich Klein approved the final manuscript.
This manuscript was handled by: David Hillman, MD.
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© 2016 International Anesthesia Research Society
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