Salidroside rescues hypoxic cardiomyocytes by regulating the EGLN1/HIF‑1α pathway
- Authors:
- Published online on: October 2, 2024 https://doi.org/10.3892/br.2024.1868
- Article Number: 180
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Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Myocardial infarction is a condition characterised by inadequate blood supply due to blockage of coronary arteries, leading to increased oxygen demand of tissues because of oxygen deficiency caused by arterial flow restriction (1). Oxygen is an essential factor that affects cell activity and the ultimate electron acceptor in the electron transport chain (ETC), making it key for cell survival. A hypoxic environment damages the ETC, limiting oxygen for mitochondrial consumption to produce ATP via oxidative phosphorylation (2,3). Hypoxia increases pulmonary arterial pressure, resulting in acute pulmonary vasoconstriction (4). Hypoxia is the primary cause of myocardial cell injury. It inhibits proliferation of myocardial cells, induces apoptosis and decreases the viability of myocardial cells (5-7).
Rhodiola rosea L., a perennial herb with various biological effects, was first used in Tibetan medicine to treat hypoxia, decrease altitude illness, lower blood pressure and improve oxygen utilisation and tolerance to hypoxia (8-10). R. rosea L. improves aerobic metabolic processes during hypoxia and exerts a protective effect on cardiomyocytes damaged by hypoxia. Salidroside (SAL), a phenolic glycoside compound isolated from R. rosea L., exhibits various pharmacological properties, including antioxidative, anti-apoptotic, anti-inflammatory and cardioprotective effects (6,11). SAL plays an important role in protecting against myocardial damage and has significant inhibitory and protective effects on damaged myocardial tissue via antioxidant production and inhibition of myocardial apoptosis (12,13). In cardiovascular disease, SAL has been reported to enhance and protect cardiac function by inhibiting cardiomyocyte degeneration, necrosis and apoptosis (14). SAL can also stimulate the accumulation of hypoxia inducible factor (HIF)-1α under hypoxia conditions, decrease hypoxia-induced injury of cells and protect cardiomyocytes from hypoxia/reoxygenation injury, indicating the potential of SAL in combating hypoxia injury (6,15,16). Furthermore, SAL alleviates cardiovascular emergency contraction caused by hypoxia, induces and improves activity of key enzymes for free radical scavenging in the body, promotes balance between free radical generation and scavenging and inhibits lipid peroxidation in the cell membrane (17-19). SAL can help resist hypoxic injury, participate in signalling pathways that antagonise hypoxic cytotoxicity and resist hypoxia-induced apoptosis by reducing the expression of associated proteins such as PI3K and AMPK, which effectively protects against ischaemic anoxic cardiovascular disease (12,13,20,21).
HIF-1 is an oxygen-regulated transcription activator that is activated because of the growth restriction of cells in hypoxia owing to blockage of oxidative phosphorylation in normal cells, consequently making glycolysis the primary energy supply mode (22). HIF-1 consists of α- and β-subunits, which are key gene regulators involved in cellular hypoxia response, erythropoiesis regulation, angiogenesis, anaerobic metabolism and glycolysis pathways (23,24). HIF-1α is a key factor that influences hypoxic response in tumour cells and is expressed cumulatively under hypoxia conditions; however, its expression levels are low under normoxic conditions (24). In addition, HIF-1α regulates cell response to hypoxia and tumour biological behaviour by influencing cell apoptosis/proliferation and energy metabolism (25).
Egl-9 family HIF 1 (EGLN1) is the primary oxygen receptor in the human body (26). Under normoxic conditions, EGLN1 uses O2 as a cofactor to hydroxylate HIF-1α. HIF-1α is recognised by the E3 ubiquitin ligase complex formed by the von Hippel-Lindau protein, resulting in its rapid degradation by proteasomes (27). However, under hypoxic conditions, activity of the proline hydroxylase EGLN1 is inhibited, which inhibits the hydroxylation and ubiquitination-based degradation of HIF-1α. This causes HIF-1α to accumulate and enter the nucleus to form a complex with HIF-1β (28). This complex regulates expression of genes downstream of hypoxia and hypoxic stress response in the body (29).
Since hypoxia-induced apoptosis of cardiomyocytes is a notable risk factor for cardiovascular disease (30,31), preventing cardiomyocyte apoptosis under hypoxic conditions is key. SAL alleviates cardiomyocyte apoptosis and enhances cell viability; however, the underlying mechanisms remain unclear. Studies on SAL have mainly focused on its ability to resist hypoxia and the mechanism underlying its anti-hypoxic effects (6,12,13); however, whether SAL mitigates existing hypoxic conditions remains to be determined. Furthermore, whether there is a regulatory interaction between EGLN1 and HIF-1α that affects apoptosis of cardiomyocytes is unknown. Therefore, the rescue ability of SAL in hypoxic H9C2 cells was studied using hypoxic culture conditions in.
Materials and methods
Materials
SAL, DMSO, trypsin, 1.5 M Tris-HCl (pH=8.8), 1.0 M Tris-HCl (pH=6.8) and penicillin-streptomycin solution were purchased from Beijing Solarbio Science & Technology Co., Ltd. Serum-free DMEM and FBS were purchased from Thermo Fisher Scientific, Inc. PBS was purchased from Lanzhou Bailing Biotech Co., Ltd. RNApure Tissue & Cell (DNase I), UltraSYBR One Step RT-qPCR, HiFiScript cDNA Synthesis and Cell Counting Kit-8 (CCK-8) were purchased from Beijing ComWin Biotech Co., Ltd. SDS-PAGE Sample Loading Buffer (5X), fluo-4/acetoxymethyl ester (Fluo-4/AM) (2 mM) and Annexin V-FITC Apoptosis Detection kit were purchased from Beyotime Institute of Biotechnology. BCA protein assay kit and 5% bovine serum albumin (BSA) were purchased from Beijing Labgic Co., Ltd. L-glutamine was purchased from Suzhou Haixing Biological Technology Co., Ltd.; 30% acrylamide (29:1) was purchased from Sinopharm Chemical Reagent Co., Ltd. PVDF membranes was purchased from MilliporeSigma. Tween-20 was purchased from Amresco Co., Ltd. Anti-EGLN1 (cat. no. #4835s) was purchased from Cell Signaling Technology, Inc. Anti-HIF-1α (cat. no. NB100-105) was purchased from Novus Biologicals, LLC. Anti-GAPDH (cat. no. 60004-1-Ig) was purchased from Proteintech Group, Inc. Horseradish peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (cat. no. ZB2301) was purchased from OriGene Technologies, Inc. 10% SDS was purchased from China Sinopharm International (Shanghai) Co., Ltd. Enhanced chemiluminescence (cat. no. ECL-0011) was purchased from Ding Guo Prosperous Co., Ltd. Primers for HIF-1α (forward, 5'-CCGCCACCACCACTGATGAATC-3' and reverse, 5'-GTGAGTACCACTGTATGCvTGATGCC-3') and GAPDH (forward, 5'-GAAGGTCGGTGTGAACGGAT-3' and reverse, 5'-CCCATTTGATGTTAGCGGGAT-3') were purchased from Beijing Tsingke Biotech Co., Ltd. EGLN1 primers (forward, 5'-AGCTGGTCAGCCAGAAGAGT-3' and reverse, 5'-GCCCTCGATCCAGGTGATCT-3') were purchased from Sangon Biotech Co., Ltd. All other solvents and chemicals were of analytical grade. Pure water was produced using a Milli-Q purification system (MilliporeSigma).
Cell culture
H9C2 cell line was purchased from Suzhou Haixing Biological Technology Co., Ltd. The cells in the normoxia group were cultured in DMEM containing 10 FBS and 1% penicillin-streptomycin and incubated at 37˚C in a 5% CO2 incubator. For the hypoxia group, cells were cultured at 37˚C with 5 CO2 and 2% O2.
In vitro cell CCK-8 assay
The biocompatibility of SAL in H9C2 cells was assessed using CCK-8 assay. H9C2 cells were seeded into 96-well plates at a density of 1x105 cells/well and incubated overnight at 37˚C, following which 200 µl SAL (10, 100, 250, 500, 750 and 1,000 nM) or DMEM (blank control) was added for an additional 24 h at 37 ˚C. After removing the DMEM, 110 µl fresh medium (containing 10 µl CCK-8) was added and incubated for 1.5 h. Finally, viability was determined by measuring the absorbance of each well at 450 nm using Spectrophotometer Multiskan (Thermo Fisher Scientific, Inc.).
The ability of SAL to rescue hypoxia-treated H9C2 cells was also confirmed using the CCK-8 method. H9C2 cells were cultured in 96-well plates at a density of 1x105 cells/well and incubated overnight at 37˚C. H9C2 cells were transferred to a hypoxic incubator and cultured under hypoxic conditions for 48 h at 37˚C. Medium was removed and 200 µl SAL (10, 100, 250, 500, 750 and 1,000 nM) or an equal volume of fresh DMEM was added for 24 h in the hypoxic incubator at 37˚C. After removing DMEM, the cells were incubated with CCK-8 solution for 1.5 h. Finally, viability was calculated by measuring the absorbance of each well at 450 nm using Spectrophotometer Multiskan (Thermo Fisher Scientific, Inc.).
Early and late apoptosis analysis
The rescue effect of SAL on hypoxic cells was quantitatively analysed using flow cytometry. H9C2 cells were co-inoculated into 3-cm dishes at a density of 2.5x105 cells/ml. A total of three groups was used: Normoxia, hypoxia and hypoxia + SAL. Cells in the normoxia group were cultured under normoxic conditions for 48 h and incubated in fresh DMEM for an additional 24 h at 37˚C. Cells in the hypoxia group were incubated under hypoxic conditions for 48 h at 37˚C, followed by incubation in DMEM for an additional 24 h. In the hypoxia + SAL group, cells were incubated under hypoxic conditions for 48 h at 37˚C, followed by incubation with 200 µl SAL (100 nM) for an additional 24 h at 37˚C. Cells were collected via centrifugation (500 x g for 5 min at 4˚C) and washed twice with PBS. Cells were suspended in 100 µl binding buffer mixed with 5 µl Annexin-V/FITC and incubated for 5 min in the dark at room temperature. The cells were mixed with 10 PI stain and 400 µl PBS. The stained cells were analyzed by flow cytometry (BD Biosciences) and analysed using WinCyte software (CompuCyte).
Intercellular Ca2+ concentration analysis
The ability of SAL to maintain intracellular calcium homeostasis was evaluated using Fluo-4/AM. H9C2 cells were cultured in 12-well plates with cell slides at a density of 1.5x105 cells/well. Normoxia group was cultured at 37˚C with 5% CO2 and 2% O2. Hypoxia group was cultured at 37˚C with 5% CO2. The culture medium was removed following incubation, the cells were washed with PBS twice and 200 µl Fluo-4/AM (2.5 µM) was added. The cells were then incubated at 37˚C for 15 min in the dark. The stained H9C2 cells were washed with PBS three times, 300 µl 4% paraformaldehyde was added to fix the cells for 30 min at 37˚C, and cells were observed under a fluorescence microscope at 100x magnification (Leica GmbH).
Tandem mass tag (TMT) proteomics analysis
A total of 1x107 cells/tube (three tubes from each group) were subjected to TMT proteomic analysis to screen differentially expressed proteins, which were analysed at the functional level as reported (32). Gene Ontology (GO; david.ncifcrf.gov/tools.jsp) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment (http://www.kegg.jp) analyses were performed to determine whether differentially expressed proteins were significantly enriched in hypoxia-related pathways. For each protein, the fold-change (FC) was calculated as the ratio of mean values of all biological measurements in each two groups.
Reverse transcription-quantitative (RT-q)PCR
RNA was extracted from cells using an RNApure Tissue and Cell kit (DNase I) according to the manufacturer's protocol. cDNA was synthesised using HiFiScript cDNA Synthesis kit according to the manufacturer's protocol. UltraSYBR One Step RT-qPCR Kit was used to detect the expression of HIF-1α and EGLN1. Thermocycling conditions were as follows: Initial denaturation at 95˚C for 30 sec, followed by 45 cycles of 95˚C for 5 sec and 60˚C for 30 sec. The relative expression levels of HIF-1α and EGLN1 were evaluated by the 2-∆∆Cq method (33).
Western blotting
Cell lysate was extracted using RIPA assay buffer and protein lysates were obtained following centrifugation (12,000 x g for 10 min at 4˚C). Proteins were quantified using a BCA assay kit. The protein extracts were isolated and transferred onto a PVDF membrane, which was blocked with 5% BSA at 37˚C for 2 h. Subsequently, the membrane was incubated overnight with primary antibody at 4˚C, followed by incubation with secondary antibody at 37˚C for 1 h. Chemiluminescent imaging system (Clinx Science) was used to detect the signals. The bands were quantified using Image J software 6.3 (imagej.net/software/).
Statistical analysis
All data were analysed by SPSS 16.0 software (SPSS, Inc.). Continuous variables are expressed as the mean ± standard deviation of three independent replicate experiments. One-way analysis of variance followed by Tukey multiple comparisons test were employed to determine significance between different groups. Fisher's exact test was used to find enriched GO and KEGG terms. The corresponding P-value was calculated as the significance index. Benjamini-Hochberg False Discovery Rate was used to correct the P-value. P<0.05 was considered to indicate a statistically significant difference.
Results
Hypoxia modelling and optimal concentration screening of SAL
Hypoxic incubator models create a hypoxic environment and induce cell apoptosis (34,35). Morphology of the adherent H9C2 cells was fusiform; however, following hypoxic culture, the cell morphology became round and some cells fell off the bottom of the culture dish (Fig. 1A). These morphological changes were not apparent in SAL treatment group. Compared with the hypoxia group, cells of the hypoxia + SAL group adhered to culture dishes and exhibited spindle-shaped shape, which was notably improved.
CCK-8 assay was used to evaluate the effect of SAL on viability of H9C2 cells under a normoxic environment (Fig. 1B). Cell viability was >90%, which confirmed good biocompatibility of SAL. The different incubation times had no significant effect on proliferation of H9C2 cells under normoxic conditions; therefore, 24 h was selected as the incubation time for SAL in subsequent experiments.
A hypoxia model was created to explore the ability of SAL to rescue H9C2 cells following hypoxia. With higher SAL concentrations, the survival rates of hypoxic cells were significantly higher than those of the control group (Fig. 1C). SAL concentrations of 100, 250, 500, 750 and 1,000 nM showed good rescue ability; therefore, 100 nM was selected for subsequent experiments.
Ca2+ detection and apoptosis
To investigate changes of intracellular Ca2+ in the three groups, the cells were stained with Fluo-4/AM and observed using a fluorescence microscope. Compared with the control group, H9C2 cells in the hypoxia group showed brighter green fluorescence, indicating that the hypoxia induced intracellular calcium overload (Fig. 2A). Following SAL treatment in hypoxic cells, green fluorescence intensity of Ca2+ was notably weakened compared with the hypoxia group. These results indicated that SAL treatment reduced intracellular calcium overload in hypoxic cells, thereby inhibiting cell apoptosis.
Flow cytometric analysis of cells stained with Annexin V-FITC/PI was used to assess the degree of apoptosis. Survival rates of H9C2 cells in the normoxia and normoxia + SAL groups were >90%, which was consistent with the cell viability results obtained by CCK-8 assay (Fig. 2B). These results further confirmed that SAL has good biocompatibility. After H9C2 cells were cultured under hypoxic conditions, ~18.45% cells were apoptotic and the apoptosis rate increased by 13.5% compared with the normoxia group, indicating that apoptosis was induced via hypoxia-related pathways following hypoxia. In hypoxic cells treated with SAL, the apoptosis rate was 8.76% (early and late apoptosis, 3.24 and 5.52%, respectively), which was 9.69% lower than that in the hypoxia group. Flow cytometry showed that SAL effectively improved survival rate of hypoxic cells and attenuated the effects of hypoxia.
Differential protein screening
According to proteomic analysis, 63 proteins were up- and 42 were downregulated in the normoxia group compared with the hypoxia group. The expression of 78 proteins was up-while that of 48 proteins was downregulated in the normoxia group compared with the hypoxia + SAL group. The expression of four proteins was up- and that of 12 was downregulated in the hypoxia group compared with the hypoxia + SAL group (Fig. S1). Subsequently, differentially expressed proteins were screened for those associated with the hypoxia pathway.
Relative protein abundance of EGLN1(974) in the hypoxia group was significantly higher than that in the control group (461) following hypoxia treatment; however, the protein abundance decreased to 737 following treatment with SAL (P<0.05; Fig. 3A). EGLN1 expression was negatively associated with oxygen concentration under hypoxic conditions.
GO and KEGG enrichment analysis
GO enriched terms were ‘response to glucose’, ‘response to hypoxia’ ‘glycolysis/gluconeogenesis’ (Fig. 3B). Therefore, cells responded to hypoxia by expressing hypoxia-related proteins; hypoxia treatment affected cellular glucose metabolism pathways. KEGG enrichment analysis showed that HIF-1 signalling pathway was enriched in the hypoxia vs. hypoxia + SAL group; seven proteins were down- and one was upregulated (Fig. 4A and B). Hypoxia affected the glycolysis/gluconeogenesis pathway, a central pathway in energy metabolism (Fig. 3C and D).
Expression of HIF-1α and EGLN1
Expression of HIF-1α and EGLN1 in the hypoxia group was significantly higher than those in the normoxia group (Fig. 5A). For HIF-1α, mRNA expression under hypoxia was 4.98-fold that of the control group, whereas treatment with SAL decreased its expression. The mRNA expression levels of EGLN1 showed a similar trend. mRNA expression of EGLN1 under hypoxia was 2.40-fold higher than that in the normoxia group and the expression of EGLN1 was reduced following co-incubation with SAL. Non-hydroxylated HIF-1α is more stable than hydroxylated HIF-1α, resulting in increased mRNA expression. EGLN1 mRNA expression increased under hypoxia.
Compared to the normoxia group, expression of HIF-1α protein significantly increased following hypoxia treatment, which was consistent with mRNA levels (Fig. 5B and C). EGLN1 protein expression increased following hypoxia treatment. However, following treatment with SAL for 24 h, expression of HIF-1α and EGLN1 protein decreased.
Discussion
Under hypoxic conditions, the ETC in the mitochondria of cardiomyocytes is blocked due to insufficient oxygen supply (36). Electrons leak out of the respiratory chain and combine with O2 to produce reactive oxygen species (ROS), causing lipid peroxidation of mitochondrial and cell membranes and ultimately apoptosis (36,37). Therefore, it is key to prevent hypoxia-induced loss of cardiomyocytes. Studies have shown that SAL exerts anti-hypoxic and protective effects on cardiac function (12,13,38). Following hypoxic stimulation in vitro, SAL treatment improves cardiomyocyte activity, protects cardiomyocytes from hypoxia-induced injury and decreases apoptosis (39). The present study further demonstrated that SAL could rescue cardiomyocytes under hypoxia, decrease apoptosis and regulate the hypoxia-associated EGLN1/HIF-1α pathway.
Cobalt chloride (CoCl2) is used to mimic hypoxic conditions because it promotes apoptosis and increases ROS production, leading to mitochondrial abnormality (40). However, CoCl2 mimics hypoxia by stabilising HIF-1α expression (41). Therefore, the present study used true hypoxia (2% O2). The present study showed that SAL could effectively relieve Ca2+ overload in hypoxic cells. Hypoxia changes cell membrane potential, leading Ca2+ to enter the cells, thereby causing calcium overload. Calcium ions play an important role in several physiological processes, such as energy production and apoptosis (42,43). Mitochondrial calcium overload induces mitochondrial swelling, mitochondrial membrane potential disorder and the release of apoptotic factors into cytoplasm, which in turn leads to apoptosis (44,45). In cardiomyocytes, mitochondria contain large calcium pools; when mitochondrial Ca2+ levels exceed their capacity, mitochondrial permeability transition pores are irreversibly opened, lowering mitochondrial membrane potential and ultimately causing apoptosis. Decreasing mitochondrial Ca2+ overload can therefore prevent cardiac injury (46). Here, SAL decreased Ca2+ concentration in cardio myocytes under hypoxic condition, which may also play a protective role.
TMT proteomics identified differentially expressed proteins associated with glucose metabolism and the hypoxia response pathway, among which EGLN1 plays an important role in hypoxia (26,27). Hypoxia inhibits activation of EGLN1, which in turn inhibits hydroxylation of the two proline residues of HIF-1α, causing HIF-1α to accumulate in the cell and promoting apoptosis. The addition of SAL alleviates effects of hypoxia environment, EGLN1 protein is activated and hydroxylates the oxygen-dependent degradation domain of HIF-1α, causing its degradation (27). SAL treatment downregulated HIF-1α and EGLN1 expression, which was increased under hypoxia. GO and KEGG analysis showed that EGLN1 protein expression was related to HIF-1α hypoxia pathway. As the upstream gene of HIF-1α, EGLN1 expression and activity directly affect the expression of HIF-1α, thus affecting apoptosis. However, SAL decreased expression of HIF-1α and EGLN1 and increased the vitality of cells.
This HIF-1α pathway is key for cellular adaptation and survival in hypoxic environments. When cells are exposed to hypoxia, HIF-1α is stabilised and dimerises with HIF-1β, which regulates the expression of genes involved in metabolism, cell proliferation and apoptosis. Several other differentially expressed proteins involved in the HIF-1 signalling pathway have been identified by TMT proteomics, including downstream metabolic regulators of HIF-1α, such as pyruvate dehydrogenase kinase (PDK1) and lactate dehydrogenase (LDHA) (47). Under hypoxic conditions, HIF-1-mediated PDK1 expression shunts glucose away from mitochondria, attenuating mitochondrial respiration and preventing toxic ROS production (48). LDHA is a key enzyme that converts pyruvate into lactic acid during glycolysis. Hypoxia-induced LDHA is reported to promote inflammation (47,49). SAL alleviates hypoxia-induced upregulation of PDK1 and LDHA, suggesting that SAL may alleviate physiological stress caused by hypoxia (47,49). The expression of HIF-1α and EGLN1 increased under hypoxia treatment; the expression of HIF-1α decreased following treatment with SAL, demonstrating that SAL decreased expression of hypoxia factors. Similarly, EGLN1 showed a consistent trend with HIF-1α. These results showed that SAL inhibited expression of hypoxia-related factors, inhibiting their effects in the signalling pathway and achieving cell rescue. SAL could effectively regulate the expression of HIF-1α and EGLN1 by regulating the EGLN1/HIF-1α hypoxia signalling pathway and inhibit apoptosis.
The GO and KEGG enrichment analysis also showed hypoxia affected the glycolysis/gluconeogenesis pathway, a central pathway in energy metabolism Gluconeogenesis refers to the process by which non-sugar substances are converted into glycogen or glucose by enzymes in organs such as the liver and kidney (50,51). HIF-1 activation by hypoxia induces glycolysis, the anaerobic oxidation of glucose, resulting in conversion of normoxic aerobic respiratory metabolic pathway to a different energy production pathway with lower oxygen consumption (50).
The rescue effect and underlying mechanism of action of SAL on cardiomyocytes remain to be confirmed in cell lines other than H9C2 cells. The concentrations required to achieve potential effects of SAL may vary in different cells; therefore, further investigation is warranted to optimize the concentration of SAL for hypoxia treatment.
In conclusion, SAL enhanced the viability of cardiomyocytes, inhibited intracellular mitochondrial calcium overload, decreased the expression of hypoxia-associated factors HIF-1α and EGLN1 and inhibited cell apoptosis through the EGLN1/HIF-1α pathway, suggesting that SAL effectively rescued the damage of cardiomyocytes caused by hypoxia (Fig. 6).
Supplementary Material
Proteomic analysis result. (A) 63 proteins were up- and 42 were downregulated in the normoxia group compared with the hypoxia group. (B) The expression of 78 proteins was up-while that of 48 proteins was downregulated in the normoxia group compared with the hypoxia + SAL group. (C) The expression of four proteins was up- and that of 12 was downregulated in the hypoxia group compared with the hypoxia + SAL group.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Natural Science Foundation of Hunan Province (grant no. 2023JJ50289) and Scientific Research Project of Hunan Provincial Health Commission (grant no. 202103011065).
Availability of data and materials
The data generated in the present study may be requested from the corresponding author.
Authors' contributions
WZ wrote and reviewed the manuscript and conceived and designed the study. ZL performed experiments and analyzed data. CX performed experiments. XL conceived and designed the study and edited the manuscript. WZ and XL confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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