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Pretreatment With Argon Protects Human Cardiac Myocyte-Like Progenitor Cells from Oxygen Glucose Deprivation-Induced Cell Death by Activation of AKT and Differential Regulation of Mapkinases

Qi, Hong∗,†; Soto-Gonzalez, Lourdes; Krychtiuk, Konstantin A.; Ruhittel, Sarah; Kaun, Christoph; Speidl, Walter S.; Kiss, Attila§; Podesser, Bruno K.§; Yao, Shanglong; Markstaller, Klaus; Klein, Klaus U.; Tretter, Verena

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doi: 10.1097/SHK.0000000000000998



Despite their reputation as inert gases, some members of the group of noble gases have been shown to exert biological effects (1, 2). The most intensively studied noble gas is the anesthetic xenon, the heaviest nonradioactive member of this group.

It is well established that xenon provides organ protection in ischemic conditions (3). One known mechanism of xenon action is antagonism of N-methyl-d-aspartate (NMDA) receptors (4). Further intracellular targets of xenon are the antiapoptotic proteins B-cell lymphoma (Bcl) 2 and Bcl-xL and mitogen-activated protein kinases (MAPKs) (5–7). A major drawback of the use of xenon is its low abundancy in the atmosphere and the respective high purification costs. Therefore, other more cost-effective noble gases could replace xenon as an organ protective agent.

Some data already exist on argon, which is a narcotic only under high pressures, showing an increased tolerance toward hypoxia and ischemia (8). In vivo models using middle cerebral artery occlusion for cerebral ischemia or left anterior descending artery occlusion for myocardial ischemia in conjunction with argon pre- or postconditioning revealed positive outcome with regards to a reduced infarct size (9, 10). Despite a reduction of cortical infarct volume, some studies report some increase in subcortical brain damage indicating a cell type specific effect (11). The mechanism of action of argon seems to be different from xenon, as argon is not a NMDA receptor antagonist. In the nervous system Fahlenkamp et al. (12) could show the activation of Erk after argon exposure.

In the present study we aimed to investigate a putative protective mechanism of argon treatment on cultured isolated human cardiac myocyte-like progenitor cells (HCMs). These cells were freshly isolated from explanted human hearts derived from heart transplantation surgery, exhibited positive staining for cardiac myocyte markers, but still proliferated before a state of terminal differentiation. We hypothesized that pretreatment with 30% or 50% argon before oxygen–glucose deprivation (OGD), a cell culture model of ischemic injury, would reduce cellular injury as assessed by lactate dehydrogenase (LDH), mitochondrial deoxyribonucleic acid (mtDNA), inflammatory cytokine release, and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay. Furthermore, we studied underlying target molecules by investigating MAPKs (extracellular signal-regulated kinase, ERK; c-jun N-terminal kinase, JNK; p38 mitogen-activated protein kinase) including downstream transcription factors and protein kinase B (Akt) that are implicated in the regulation of apoptosis and in prosurvival pathways, respectively.


Isolation and culture of HCMs

Ethical approval for this study was provided by the Medical University of Vienna Ethics Committee, Vienna, Austria, on May 6, 2015 (chairperson: Professor E. Singer). Experiments were performed after obtaining written informed consent from patients between July 2015 and December 2016. Primary cultures of HCMs were prepared from ventricular tissue obtained from explanted hearts from three patients undergoing heart transplantation for severe ischemic or dilative cardiomyopathy as described (13, 14).

Ventricular tissue was cut into small pieces in phosphate-buffered saline (PBS) pH 7.4 without enzymatic digestion (PBS; Gibco, Invitrogen, Carlsbad, Calif). The cell mass was filtered through a 40 μm cell strainer and centrifuged at 1,200 rpm for 10 min. The pellet was washed twice with PBS. Cells were resuspended in Dulbecco's modified eagle medium (DMEM) containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin, plated into a Petri-dish and incubated for 60 min at 37°C with 5% carbon dioxide (CO2) in the atmosphere to separate myocytes from fibroblasts by preplating. The nonattached cells were centrifuged and rinsed twice with PBS. The cell pellet was resuspended in DMEM containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL transferrin, and 10 μg/mL insulin. HCMs were seeded into fibronectin-coated flasks at a density of 104 cells/cm2. Cultured HCMs were characterized by staining for troponin I, tropomyosin, cardiotin, and myocardial muscle actin.

Only cultures in which >95% of the cells stained positive for cardiac myocyte markers (troponin I, tropomyosin, cardiotin, and myocardial muscle actin) were used in this study. In these cultures, contamination with smooth muscle cells, endothelial cells, and fibroblasts as judged by staining for smooth muscle actin, von Willebrand factor, and fibroblast specific antigens was <2% (11). All in vitro experiments were performed with HCMs at passage 2 to 4. The harvesting and maintenance of HCMs were described before experimentation (15). Briefly, HCMs were recovered in a 1% gelatin-coated (Sigma-Aldrich, St. Louis, Mo) 75 cm2 culture flask (Falcon, BD Biosciences, Schwechat, Austria) and maintained in Medium 199 (M199; Gibco, Invitrogen, Carlsbad, Calif) supplemented with 20% heat-inactivated fetal bovine serum (FBS superior; Biochrom, Berlin, Germany), 10 mM l-glutamine, and 1% penicillin/streptomycin (Gibco, Invitrogen) under a humidified atmosphere containing 5% CO2 at 37°C.

Oxygen–glucose deprivation

For the experiments cells were used at passage 2 to 5 and seeded into 24 well plates with gas permeable membranes at the bottom (Zellkontakt, Nörten-Hardenberg, Germany) at a density of 3.104 cells per well in M199 medium containing 20% fetal calf serum supplemented with l-glutamine, penicillin, and streptomycin. For argon gas exposure plates were placed into boxes with 24 gas-outlets at the bottom that were connected to premixed gas bottles containing mixtures of 30% or 50% argon, 21% oxygen, nitrogen, and 5% CO2. OGD was performed by changing the medium to basal DMEM medium without glucose and serum immediately after argon application and exposing the cells to 95% nitrogen and 5% CO2 for 16 h (Appendix 1, For reperfusion, the medium was replaced with full medium containing glucose and 20% fetal calf serum and cells were incubated in a standard incubator at 37°C, 5% CO2, and otherwise ambient atmosphere.

Analysis of apoptotic states

After argon gas exposure and OGD, cells were trypsinized, stained with fluorescein isothiocyanate-labeled annexin V and propidium iodide, and analyzed by flow cytometry (FC500 Beckman Coulter, Indianapolis, Ind). The method allows the analysis of different apoptotic and necrotic states: healthy cells (annexin V/PI negative cells), early apoptosis (Annexin V positive/PI negative cells), late apoptosis (Annexin V positive/PI positive cells), and necrotic cells (Annexin V negative/PI positive cells). For the analysis of pathway specific effects cells were incubated with the MEK1 inhibitor U0126 (10 μM), the Akt Inhibitor MK2206 (1 μM), and the JNK inhibitor SP600125 (10 μM) 30 min prior and during argon exposure (all substances were from Tocris Bioscience, Bristol, UK). Stock solutions of inhibitors contained DMSO as solvent, and the final DMSO concentration in the cell culture medium was not more than 0.1%. Control samples were incubated with the same amount of DMSO without inhibitor. Further controls were analyzed that contained the respective inhibitors without argon exposure.

TUNEL assay

The TUNEL assay was used as an in situ test for apoptosis (Click-iT TUNEL Alexa Fluor imaging assay; Invitrogen, ThermoFisher Scientific, Waltham, Mass). The principle of the assay is the incorporation of fluorescently modified dUTPs by the enzyme terminal deoxynucleotidyl transferase at the 3’-OH ends of fragmented DNA that appear in the course of the apoptotic process. Counterstaining for total nuclei was performed using Hoechst 33342 staining.

LDH and mtDNA analysis

LDH is a cytosolic enzyme, that is released, when the cell membrane becomes leaky in the course of apoptotic or necrotic events. Therefore, LDH release can be used as a quantitative measure for cell deterioration. We used the cytotoxicity detection kit (Roche Diagnostics GmbH (Schweiz), Basel, Switzerland).

DNA copy numbers in the cell culture supernatants were measured by quantitative real-time polymerase chain reaction for the mitochondrial genes cytochrome b, cytochrome c oxidase subunit III, and nicotinamide adenine dinucleotide dehydrogenase using LightCycler TaqMan Master (Roche) according to manufacturer's instructions. Cycling conditions were the following: 10 min at 95°C followed by 60 cycles of 95°C for 15 s and 60°C for 30 s. Primers for cytochrome b (forward 5’-CATCTTGCCCTTCATTATTGC-3’, reverse 5’-GTTGTTTGATCCCGTTTCGT-3’), cytochrome c oxidase subunit II (forward 5’-CAAACATCACTTTGGCTTCG-3’, reverse 5’-AGTCAAACCACATCTACAAAATGC-3’), and NADH dehydrogenase (forward 5’-CTACCGCATTCCTACTACTCAACTT-3’, reverse 5’-GCTTGTTTCAGGTGCGAGAT-3’) were designed using the Roche Universal Probe Library Assay Design Centre (

ELISA analysis

The release of different cytokines such as interleukin (IL-)1β, IL-6, IL-8, and vascular endothelial growth factor (VEGF) was monitored using the human Duoset ELISA (R&D Systems, Minneapolis, Minn).

Westernblot analysis

Cells were exposed to standard air or a 30% argon mixture for 0, 15, 30, 45, 60, or 90 min, washed with ice-cold phosphate buffered saline, and lysed in RIPA buffer (50 mM Tris, 150 mM NaCl pH 7,6, 1% Triton X100, 0.5% sodium deoxycholate, 0,1% sodium dodecyl sulphate, protease, and phosphatase inhibitor mini tablet (Pierce)). After protein precipitation, equal amounts (50 μg total protein) of protein were applied to SDS-polyacrylamid gel electrophoresis and blotted onto nitrocellulose membranes. Membranes were blocked with 5% BSA in Tris-buffered saline/0.1% Tween 20 and incubated with primary antibodies over night at 4°C (antigens: ERK1/2, phospho(Thr202/Tyr204)-ERK1/2, JNK, phospho(Thr183/Tyr185)-JNK, Akt, phospho(Ser473)-Akt, p38, phospho(Thr180/Tyr182)-p38 antibodies from Cell Signalling Technologies, Danvers, Mass; dilutions as suggested by the company). Finally, blots were developed using alkaline phosphatase-conjugated secondary antibodies and CDP-star reagent (Sigma-Aldrich) followed by exposure to chemiluminescence film (Hyperfilm; GE Healthcare, Pittsburgh, Pa) and quantification by densitometry.

Analysis of MAPK transcription factor activity

Downstream activation of transcription factors by MAPkinases was analyzed for c-Jun, ATF2, c-Myc, MEF2, and STAT1alpha by a colorimetric test kit from Abcam. A double-stranded DNA sequence containing the MAPK consensus binding site is coated in a microtiter plate. Activated transcription factors bind to this sequence and are recognized by an antibody specific for the activated form. The assay is finally developed with an HRP-conjugated secondary antibody and TMB as substrate.

Analysis of Akt activity

Akt activity was measured using a colorimetric assay (Abcam). The kit contains a microtiter plate with an immobilized synthetic peptide that functions as a substrate for phosphorylation by Akt kinase. The phosphorylated peptide is detected and quantified by an ELISA.

Statistical analysis

Experiments were performed in triplicates three times in total, whereby cultures were from three different donors. Data were analyzed for Gaussian distribution using the Kolmogorov Smirnov normality test. Data were expressed as mean ± SD. Means were compared either by Student t test (two groups) or by two-way analysis of variance (ANOVA) for independent samples with pair-wise Bonferroni post hoc tests for more groups. If not indicated otherwise, adjusted P values are considered significant at a P < 0.05. However, as the study was conducted in an explorative approach, all P values are considered as explorative. Statistical analysis and data plotting were performed using Prism 6.0 software (GraphPad, San Diego, Calif).


Argon protects HCMs from apoptosis and necrosis

Procedures and all analysis are schematically depicted in the study protocol (Appendix, We used OGD to induce apoptosis and quantified apoptotic and necrotic states by Annexin V/propidium iodide staining and flow cytometry analysis (Fig. 1A shows representative plots for all treatments; Fig. 1B summarizes quantifications from three individual experiments). Sixteen hours of OGD reduced the portion of healthy cells from 85.3 ± 1.8% to 47.6 ± 3.0% (P < 0.0001 vs. control). Preincubation with a mixture of 30% argon for 90 min before OGD significantly increased the portion of healthy cells to 67.2 ± 0.7% of total cells (P < 0.0001 vs. OGD). Furthermore, argon-treated samples showed a decrease in both early apoptosis (9.9 ± 5.4% vs. 18.8 ± 4.4%; P < 0.01 vs. OGD) and late apoptosis (14.5 ± 2.4% vs. 25.9 ± 3.2%; P < 0.0001 vs. OGD), but no difference in necrosis (8.3 ± 3.9% vs. 7.4 ± 3.1%). Similarly, pretreatment for 90 min with 50% argon increased the percentage of healthy cells to 69.3 ± 3.0% with 8.7 ± 0.7% of cells in early apoptosis, 10.3 ± 1.2% of cells in late apoptosis, and 11.7 ± 2.4% in necrotic cells.

Fig. 1:
Cytoprotective effects of argon preconditioning against OGD-induced apoptosis.

The protective effect by argon exposure was blocked in the presence of inhibitors for the ERK or Akt pathway, and, to a lesser extent also, for the JNK pathway (Fig. 1). U0126, a MEK1 (ERK pathway) inhibitor, decreased the portion of healthy cells (53.6 ± 1.6% vs. 67.2 ± 0.7%; P < 0.001 vs. argon 30%) with 8.0 ± 1.1% early apoptotic, 24.2 ± 2.5% late apoptotic, and 14.3 ± 2.3% necrotic cells. MK2206 reduced the portion of healthy cells to 43.1 ± 0.6% (P < 0.0001 vs. argon 30%) with 14.8 ± 0.6% early apoptotic, 23.0 ± 1.2% late apoptotic, and 19.1 ± 2.3% necrotic cells. Inhibition of the JNK pathway by SP600125 resulted in 57.5 ± 4.3% healthy cells (P < 0.0001 vs. control), 2.6 ± 0.6% early apoptotic, 16.8 ± 0.8% late apoptotic, and 22.8 ± 3.9% necrotic cells. A potential direct toxic effect of kinase inhibitors was ruled out by analysis of HCMs that had been exposed to inhibitors for the same amount of time in the absence of argon (control + inhibitors).

The antiapoptotic effect of argon treatment could also be demonstrated by TUNEL staining, visualizing the double DNA strand breaks that occur in the course of apoptosis (Fig. 2). The number of highlighted nuclei is markedly reduced, when HCMs were pretreated with argon compared with OGD alone.

Fig. 2:
Reduction of apoptotic events after preconditioning with argon.

MAPkinase and Akt activation

Westernblot analysis was used to assess the phosphoactivation of MAPKs and Akt. HCMs were exposed to argon for different durations of time: 0, 15, 30, 45, 60, and 90 min. Phosphorylation of Akt on serine-473, the downstream target of mTOR, was increased after 30 min and persisted until 90 min, whereas phosphorylation of threonine-308, the downstream target of PDK-1 did not show any changes (not shown). JNK was acutely activated after 15 min, but was dephosphorylated thereafter to a level below baseline. ERK1/2 phosphorylation slightly increased after 15 min and further after 45 min compared with baseline. Phosphorylation of p38 did not show a clear tendency in intensity (Fig. 3, A and B).

Fig. 3:
Activation of Akt and MAPkinase pathways by argon preconditioning.

We further assessed downstream effects of MAPkinases activity. The transcription factor c-jun, a member of the AP-1 family, showed a strong increase in activation as early as 15 min until after 90 min of exposure to 50% argon. The transcription factors ATF2, c-myc, MEF2, and STAT1α started at a lower level, but similarly all increased after 15 min and with the exception of c-myc remained elevated until 90 min of exposure to 50% argon (Fig. 3C). Furthermore, an enzymatic Akt kinase activity assay was performed and revealed an increase of phosphorylation capacity as soon as 15 min after the start of argon exposure (Fig. 3D).

Release of LDH and cytokines

Argon preconditioning improved the cell integrity as shown by LDH and mtDNA release (Fig. 4). LDH and mtDNA in the supernatant were significantly higher in cultures that had not been incubated with an argon atmosphere before OGD (P < 0.05). Accordingly, it could be observed that HCMs secreted less inflammatory cytokine IL-1β especially during the reperfusion phase after OGD, when they had been pretreated with argon (P < 0.01) (Fig. 5). IL-8 release was reduced during argon treatment; IL-6 remained unchanged. There was a tendency of higher VEGF release during reperfusion when cells were pretreated with argon (P < 0.05).

Fig. 4:
Protective effects of argon preconditioning as assessed by cell integrity markers.
Fig. 5:
Effect of argon preconditioning on cytokine release of HCMs after OGD and reperfusion.


We investigated the effects of treatments with the noble gas argon on OGD-induced oxidative injury in human cardiac myocyte-like progenitor cells. Preexposure to a normoxic 30% and 50% argon mixture for 90 min resulted in a significant increase in the number of viable cells and a concomitant reduction in both early and late cellular apoptosis. These findings could be confirmed by TUNEL assay. The effect was reversed by the application of MAPK and Akt inhibitors during argon exposure. Argon treatment significantly reduced release of LDH, mtDNA, and IL-1β, and was significantly mediated via the ERK1/2, JNK, and Akt pathway. It cannot be excluded that other antiapoptotic mechanisms also play a role, which have not been investigated in this study. An example could be miRNAs that have been shown to play a role in the protective effects of the noble gas xenon in septic acute kidney injury (16). The present in vitro findings provide evidence that myocardial protection against oxidative stress-related injury by conditioning with argon is at least partly mediated by phosphoactivation of MAPK and Akt pathways. With regard to this argon lines up as a candidate in a row with other protectant medical gases like xenon or hydrogen sulphide (H2S) previously shown to exert antioxidant, antiapoptotic, and anti-inflammatory properties (1, 12).

Argon preconditioning protects HCMs

The noble gas argon has only anesthetic properties under hyperbaric conditions and has been mainly investigated for its neuroprotective effect. The underlying mechanism was shown to include upregulation of ERK1/2 via MEK in neurons and glia cells (12). In 2007, Pagel et al. (10) were the first to investigate the protective effects of the noble gases argon, helium, and neon against ischemia–reperfusion injury in rabbit hearts. The authors concluded that noble gases without anesthetic properties produce cardioprotection by activating prosurvival signaling cascades and inhibiting mitochondrial permeability transition pore opening in rabbits.

Our data obtained from HCMs confirm that argon also works in human cells and activates JNK, ERK1/2, and Akt pathways, thus contributing to the understanding of translation of experimental data to the clinical setting. The beneficial effects of other noble gases like xenon have primarily been shown in small rodents or rabbits. However, similar dosage regimens have been shown to be ineffective in pigs or required application of xenon prior and during ischemia (17, 18). In the present in vitro study investigating cardioprotection by argon, we could show that application of 30% argon significantly increased viability of human myocardial cells by 20%. Furthermore, argon 30% decreased release of LDH by 33%, mtDNA by 45%, and IL-1β by over 90% during reperfusion. The present study results are comparable with the effect of other noble gases such as xenon. The mechanisms of protection by xenon have not been characterized into every detail, but different studies have shown the involvement of PKC-ε, MAPkinases, and PI3kinase as well as cytoskeletal-associated factors and the mitochondrial KATP channel (19–22). PI3kinase is an upstream effector of Akt that has been shown to promote argon-mediated protection of cardiomyocytes in our model, though it seems that Akt is activated here through a different pathway. ERK1/2 and Akt are part of the reperfusion injury salvage kinase (RISK) pathway that has been indicated to play a role in the myocardial protection against ischemia reperfusion injury in ischemic and anesthetic pre- and postconditioning (23, 24). It is of note that treatment with xenon reduced infarct size to a similar degree as ischemic and anesthetic (isoflurane) preconditioning, which are established measures of cardioprotection in the perioperative setting. Also, the mechanisms seem to reveal some analogies. Pagel et al. (10) have also shown that helium can induce cardioprotection in a rabbit model of myocardial infarction with PI3kinase, ERK1/2, and p70S6kinase inhibitors reversing the effect. Therefore, the mechanisms by which the noble gases exert their protective effect seem to at least partially overlap. A significant difference seems to be the activation of c-jun N-terminal kinase (p46/p54 MAPK, JNK). Weber et al. tested the involvement of this MAPkinase in xenon-induced reduction of infarct size. The specific inhibitor SP600125 did not abolish the protective effect, neither did xenon preconditioning alter the phosphorylation state of the kinase itself (25). Our study shows that argon rapidly activates the phosphorylation of JNK within 15 min and subsequently again dephosphorylates the protein to below basal levels. Interestingly, the JNK inhibitor SP600125 reduces the protective effect of argon on cardiomyocytes, albeit to a lesser extent than the MEK1 inhibitor U0126. We also measured downstream effectors of MAPkinase activation. c-Jun, a member of the activator protein-1 (AP-1) family of transcription factors, is activated by ERK1/2 and JNK pathways and is involved in proliferation and cell cycle progression. c-jun activity is strongly upregulated by argon treatment of HCMs. Previous work indicated that deficiency in c-jun is associated with increased rate of cardiomyocyte apoptosis (26). ATF-2 is a member of the ATF/CREB family of transcription factors, binds to the cAMP response element (CRE), and is activated by ERK1/2, JNK, and p38. STAT1 is supposed to be activated by p38 and JNK pathways, and MEF2 is activated by ERK5 and p38 pathways. c-myc is an ERK substrate and regulates cell growth, differentiation, and apoptosis. All of these latter transcription factors showed an increase in activity, albeit to a smaller degree than c-jun. Activation of Akt occurred via phosphorylation of Ser473 and the Akt inhibitor MK2206 could fully reverse the protective effect of argon. The small decrease of viability of HCMs (control) in the presence of MK2206 is not statistically significant and is far less pronounced than the effect of this inhibitor on argon preconditioning before OGD. Therefore, it can be assumed that this effect is due to a real reversal of activated Akt signaling during argon exposure. A slight increase of Akt kinase activity could also be shown by using a synthetic Akt substrate.

The impact of xenon on cytokine levels was investigated in a porcine model with coronary artery occlusion showing a reduction of plasma levels of tumor necrosis factor alpha and IL 6 (18). We show that argon can reduce the release of the inflammatory cytokine IL-1β by HCMs during ischemia and reperfusion. IL-6 and IL-8 are constitutively released by HCMs in culture independent of OGD. This might be a reason, why these cytokines are less affected by argon exposure. Generally, a reduced release of inflammatory cytokines like IL-1β might favorably affect other cells in the myocardium like endothelial cells to inhibit the interactions with neutrophils.

An important issue represents the noble gas dosage. Our results in this study were obtained with lower concentrations of argon (30% and 50%) than reported in the literature (70%), showing that a broad concentration spectrum seems to be effective. This would be helpful if argon was to be used in the clinics as it offers a larger therapeutic window including higher oxygen levels. But an optimal dosage regimen is still missing. Pagel et al. (27) demonstrated that repeated cycles of helium administration progressively increased the reduction in infarct size in rabbits. In preliminary experiments performed before the present study, we also have tried this approach. However, unfortunately, we were not able to show any cell protection with short cycles of argon application similar to traditional preconditioning protocols in vitro. We propose that both dosage and timing of noble gas application are critical to obtain an optimal cardioprotective effect. Clearly, a constant low dose (30% or 50%) of argon can be administered much easier in the clinical setting.

Clinical considerations

The incidence for perioperative myocardial infarction ranges about 8% in patients over the age of 45 years. Mortality is 10% at 30 days after surgery and about twice as high as in a nonoperative setting. Different pharmacological strategies have been investigated how to reduce perioperative myocardial infarction, including avoidance of nitrous oxide and administration of beta-blockers, aspirin, or clonidine. However, a safe and effective way to prevent or reduce perioperative myocardial infarction remains unknown. As highlighted above, preconditioning of the myocardium only routinely is performed in cardiac surgery by using volatile anesthetics (e.g., isoflurane, sevoflurane, desflurane) for some time period (28). However, such strategy cannot be performed when the patients remain awake. A novel simple, safe, and effective pharmacologic approach is needed that can be administered in the awake patient. In contrast to xenon or hydrogen 2%, inhalation of argon could be such a novel approach as argon is nontoxic, non-narcotic, cheap, everywhere available and safely administrable solely by inhalation. Our results add to the literature demonstrating that argon can mediate protection not only in the brain but also in the myocardium. The authors suggest that the present in vitro results in primary human myocardial cells should be extended in in vivo studies before this approach should be investigated in human subjects. A better dose–response and time response for myocardial preconditioning must be investigated before a human study. In summary, results, however, seem promising and argon theoretically could be administered in every patient—even at minimal risk for myocardial injury—in the perioperative period. Particularly important to note is that argon could also be given preclinically or during the time of intervention and that no mechanical ventilation is required for inhalational application of argon.


This study has several limitations. An important issue is the cell type used in this study. We have been using freshly isolated cells from human heart that develop into mature cardiac myocytes after an extended period of time in culture. These cells exhibit staining for differentiated cardiac myocytes but are still proliferating to some degree, a reason why we call theses cells human cardiac myocyte-like progenitors. Vulnerability and signaling in response to hypoxic states must be assumed to be altered in developing heart cells (29), but the protective effect of argon on these cells is indisputable. A further issue is the fact that these cells have been isolated from sick individuals with ischemic/dilated cardiomyopathy and might therefore have undergone changes in structure (hypertrophy), metabolism (for instance, a shift in utilization of different energy substrates), gene expression (induction of genes normally expressed during development), and signaling, and might therefore exhibit different responses to hypoxic exposure. As healthy human hearts are usually not available for cell culture experiments, in vivo trials using small animals should be extended to investigate the signal transduction pathways involved in argon conditioning in healthy hearts in further detail. Furthermore, we did not evaluate a full dose–response and time response including delayed application of argon (postconditioning) but planned our study according to previous cell culture studies investigating neuroprotection by argon. We suggest that cardioprotection should be investigated in more detail in in vivo settings using small animals, including the conditioning effect of argon during reperfusion. Such scenario would mimic the clinical situation when argon could be given during the time of interventional therapy after myocardial infarction. It would have been very interesting to better establish a potential upstream receptor involved in the argon response. It is still not known, whether noble gases initially interact with cell surface receptors or alternatively passively diffuse through the cell membrane and meet their specific targets in the intracellular space. G-protein-coupled receptors have been discussed as candidates, but other cell surface receptors that activate prosurvival signaling kinases might be involved likewise. It would also be interesting if intermittent argon exposure might show a more pronounced effect on myocardial preconditioning.


A normoxic 30% and 50% argon mixture applied for 90 min before OGD exerts significant and equivalent protective effects against apoptosis and inflammation in HCMs. These effects seem to be mediated at least partly by the activation of JNK, ERK1/2, and Akt pathways. Cardiac myocytes are the principal cell type of the human heart. Our findings warrant more detailed studies also in other myocardial cell types and in vivo models of myocardial ischemia to assess the clinical potential of argon treatment for patients at risk for myocardial injury.


This study was presented as a poster at the Joint Meeting on Vascular Biology, Inflammation and Thrombosis, March 31 and April 1, 2016, Medical University of Vienna, Austria. This project was financially supported by funds of the major of Vienna. We thank Prof. Andreas Spittler for his advice on flow cytometry analysis.


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Apoptosis; argon; human cardiac myocyte progenitor cells; inflammation; preconditioning

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