Klotho protein contributes to cardioprotection during ischaemia/reperfusion injury

Abstract Restoration of blood flow to ischaemic heart inflicts ischaemia/reperfusion (I/R) injury, which manifests in metabolic and morphological disorders. Klotho is a protein with antioxidative and antiapoptotic activity, and is involved in the regulation of inflammation and fibrosis. The aim of the current research was to determine the role of Klotho in the heart subjected to I/R injury, as well as to study Klotho as a potential cardioprotective agent. Human cardiomyocytes and Wistar rat hearts perfused using Langendorff method subjected to I/R have been used. Hemodynamic parameters of heart function, markers of I/R injury, and gene and protein expression of Klotho were measured. Human cardiomyocytes were also incubated in the presence of recombinant Klotho protein, and the viability of cells was measured. There was a higher expression of Klotho gene and protein synthesis in the cardiomyocytes subjected to I/R injury. The compensatory production and release of Klotho protein from cardiac tissue during I/R were also shown. The treatment of cardiomyocytes subjected to I/R with Klotho protein resulted in increased viability and metabolic activity of cells. Thus, Klotho contributes to compensatory mechanism during I/R, and could be used as a marker of injury and as a potential cardiopreventive/cardioprotective agent.


| INTRODUC TI ON
The standard procedure for treatment of myocardial infarction (MI) is coronary reperfusion. 1,2 Whereas reperfusion is mandatory in the therapeutic approach to ischaemia, revascularization and restoration of the blood flow to ischaemic myocardium inflicts additional damage-ischaemia/reperfusion (I/R) injury. In turn, I/R injury causes metabolic, morphological and contractile disorders, leading to irreversible microvascular damage or myocardial stunning. 3,4 An excessive formation of reactive oxygen species (ROS), degradation of contractile proteins by proteolytic enzymes and necrotic cell death are important factors contributing to the pathogenesis of I/R injury. 3,5 Klotho (KL) is a membrane-bound or soluble antiaging protein with antioxidative and antiapoptotic activity. The expression of Klotho gene is observed mainly in the kidneys and brain. 6,7 It was proved that soluble KL is involved in the regulation of oxidative stress, inflammation and fibrosis. [8][9][10][11] Experimental studies of | 6449 OLEJNIK Et aL.
exogenous Klotho protein administration have apparently affirmed its protective role in several cell and animal models. 10,[12][13][14] In favour of a rationale that supports the protective role of Klotho, we decided to examine Klotho as a potential preventive/therapeutic agent in I/R heart injury.
The aim of this study was to investigate the role of Klotho protein in the heart tissue subjected to I/R injury, as well as to study Klotho as a potential cardioprotective agent.

| MATERIAL S AND ME THODS
In this study, animal tissues obtained from our other project were

| The protocol of in vitro chemical I/R injury of cardiomyocytes
Cardiac myocytes in culture underwent in vitro chemical I/R in accordance with the guidelines for experimental models of myocardial ischaemia and infarction. 15 The scheme of the experimental protocol is shown in Figure 1. Briefly, HCM underwent 15 minutes of aerobic stabilization, 15 minutes of in vitro chemical ischaemia and 20 minutes of reperfusion, 16 in the presence and absence of 1 µg/ mL 17 16 The optimal duration of ischaemia (15 minutes) was established experimentally by measurement the activity of lactate dehydrogenase (LDH) released from cells as a marker of cell injury (data not shown). In the I/R group, after 15 minutes of aerobic stabilization in HEPES buffer at RT, the buffer was removed by centrifugation (1 minute 1500 g) and the cell pellet was resuspended in ischaemia buffer and incubated for 15 minutes at RT. Then, the cells were centrifuged for 1 minutes at 1500 g, the buffer was removed and cells were incubated in aerobic conditions with HEPES buffer for 20 minutes at RT (reperfusion). After reperfusion, the buffer was removed by centrifugation at 1500 g for 5 minutes, and the cell pellet was homogenized.
The cells from aerobic control group were incubated aerobically for 50 minutes in HEPES buffer at RT. In the Klotho experimental groups (aerobic + Klotho, I/R + Klotho), the cells underwent experimental F I G U R E 1 Experimental protocol for in vitro chemical IR injury of cardiomyocytes with and without Klotho administration. I/R, ischaemia/reperfusion protocol in the presence of Klotho protein (1 µg/mL final concentration) during whole procedure. Myocytes from aerobic group subjected to Klotho protein were tested to check the cytotoxicity of Klotho.

| Cell homogenization
Cells underwent three cycles of freezing (in liquid nitrogen) and thawing (at 37°C) in the homogenization buffer (50 mmol/L Tris-HCl [pH 7.4] containing 3.1 mmol/L sucrose, 1 mmol/L DTT, 10 µg/mL leupeptin, 10 µg/mL soybean trypsin inhibitor, 2 µg/mL aprotinin and 0.1% Triton X-100). Then, myocytes were homogenized mechanically on ice (three times for 10 seconds) with a hand-held homogenizer. Supernatants were obtained by centrifugation at 10 000 g for 5 minutes at 4°C and then were transferred into a fresh tube. The homogenates were stored at −80°C for further experiments.

| Klotho mRNA expression
Total RNA from HCM was isolated with TRIZOL reagent (Thermo

| Klotho protein concentration in cardiomyocytes
Klotho protein concentration in cell homogenates was measured quantitatively using Sandwich Human Klotho ELISA Kit from Biorbyt (Biorbyt Ltd.), according to manufacturer's instruction. After resetting the blank well, the optical density (OD) (at 450 nm) of each well was measured by Spark multimode microplate reader (Tecan Trading AG). Klotho protein concentration was normalized to total protein concentration in cell homogenates and expressed in pg/μg protein.
The concentration of Klotho was compared in cells that were exposed to aerobic conditions and cells subjected to I/R.

| Immunofluorescence staining of cardiomyocytes
The cells were cultured in 24-well cell culture plate (Greiner Bio-One of Klotho was compared in cells that were exposed to aerobic conditions and cells subjected to I/R.

| Cytotoxicity assay for cardiomyocytes
To assess an influence of Klotho protein on cell viability, the CytoTox-Glo™ Cytotoxicity Assay (Promega) was performed. 18 The assay uses a luminogenic peptide substrate to measure 'dead-cell protease activity', which is released from cells that have lost membrane integrity. The assay selectively detects dead cells, because substrate cannot cross the intact membrane of live cells and does not generate signal from the live-cell population. In brief, cells were seeded in 96-well plate at a density of 1 × 10 4 cells/well for 24 hours and then subjected to in vitro chemical I/R injury in the presence and absence of 1 µg/mL Klotho protein (please see the protocol shown in Figure 1). The luminescence was recorded on Spark multimode microplate reader (Tecan Trading AG). The data were normalized to cell confluence and expressed in relative light units (RLU). The viability of myocytes was based on the number of death cells in each well and compared to cells that were exposed to aerobic conditions and cells subjected to I/R and I/R with the addition of recombinant Klotho.

| The metabolic activity of cardiomyocytes
The metabolic activity of cells subjected to I/R was assessed using the vital inclusion dye fluorescein diacetate (FDA) and the vital exclusion dye DAPI. 19 In not injured cells, appropriate esterases are present in an active form. Viable cells have the capability to incorporate the non-polar, non-fluorescent FDA and rapidly hydrolyse it into fluorescein using acetyl esterase. Fluorescein is a polar, fluorescent compound which is retained within the cell. In the damaged cells, esterified fluorescein is rapidly diffuse from cell to liquid medium and cell visualization is not allowed. Non-viable cells are susceptible to DNA staining with DAPI, where fluorescence is produced by interacting with the nucleus. On the basis of these principals, FDA can be easily used in a double staining procedure in combination with DAPI. 20,21 Briefly, myocytes were seeded in 96-well plate at a density of 2 × 10 4 cells/well for 24 hours and then subjected to in vitro chemical I/R injury in the presence and absence of 1 µg/mL Klotho protein (please see the protocol shown in Figure 1).
The cells were washed three times with PBS and stained with 5 μg/ mL FDA (Sigma-Aldrich) and 1:1000 DAPI (Sigma-Aldrich) for 15 minutes in the dark. ZOE Fluorescent Cell Imager (Bio-Rad) was used to estimate the bright green fluorescence for FDA and blue fluorescence for DAPI. Cells were considered metabolically active when stained with FDA and excluded DAPI, whereas cells that excluded FDA and stained with DAPI were considered necrotic. 19 Image J 1.52a software (NIH) was used to analyse the area of fluorescence of each image. To determine the metabolic activity of cells, green fluorescence (live cells) was normalized to the total number of cells (green + blue fluorescence) in each experiment. 19 Data were collected from three independent images trials for each experiment and shown as AU. The metabolic activity was compared in cells that were exposed to aerobic conditions and cells subjected to I/R and I/R with the addition of recombinant Klotho.

| Isolated rat hearts perfused with the Langendorff method
To determine the expression of Klotho protein at the tissue level, isolated rat heart model was used. Ten-to 11-week-old male Wistar rats (250-350 g) were treated with buprenorfin (0.05 mg/kg, ip) and After stabilization, the hearts were subjected to protocol of global I/R injury. As haemodynamic end-points of cardioprotection, 22 coronary flow (CF), heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (PED) and intraventricular pressure (dp/dt) were determined using an EMKA recording system with IOX2 software (EMKA Technologies). At the end of protocol, isolated hearts were immediately immersed in liquid nitrogen and stored at −80°C before further investigations.

| Global ischaemia/reperfusion of isolated rat hearts
Hearts in I/R group were subjected to aerobic perfusion for 25 minutes. After oxygenation, the heart perfusion was stopped (by cessation of the buffer flow) and hearts underwent global no-flow ischaemia for 22 minutes. 16 Then, hearts were perfused in aerobic conditions for 30 minutes (reperfusion). The hearts from aerobic control group were perfused aerobically for 77 minutes. At the beginning of reperfusion (47 minutes), 15 mL of coronary effluents was collected for biochemical studies. To determine cardiac mechanical function, the recovery of rate pressure product (RPP) was expressed as the product of HR and LVPD and evaluated at 25 minutes of experiment (the end of aerobic perfusion) and at 77 minutes (the end of reperfusion). Then, the analysis of functional and biochemical changes between aerobic and I/R group was performed.

| Preparation of heart homogenates
Hearts previously frozen at −80°C were crushed using a mortar and pestle in liquid nitrogen and then homogenized in ice-cold homogenization buffer containing: 50 mmol/L Tris-HCl (pH 7.4), 3.1 mmol/L sucrose, 1 mmol/L dithiothreitol, 10 mg/mL, leupeptin, 10 mg/mL soybean trypsin inhibitor, 2 mg/mL aprotinin and 0.1% Triton X-100. The homogenate was centrifuged at 10 000g at 4°C for 15 minutes, and the supernatant was collected and stored at − 80°C for further experiments.

| Qualitative and quantitative analysis of Klotho protein in the heart tissue and coronary effluents
The expression of Klotho protein in the rat heart tissue was deter-

| Assessment of LDH level
To determine the activity of LDH in coronary effluents, Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich) according to manufacturer's instruction was used. LDH is a stable cytosolic enzyme that is released upon membrane damage/permeability or cell lysis and serves as a marker of cell damage. LDH level was normalized to CF and compared in coronary effluents of hearts that were exposed to aerobic conditions and hearts subjected to I/R.

| Assessment of the number of death cells in rat hearts
To assess an influence of I/R injury on isolated rat hearts, the number of death cells using the CytoTox-Glo™ Cytotoxicity Assay (Promega) according to manufacturer's instruction was tested. The measurement of extracellular activity of dead-cell protease was performed in coronary effluents and normalized to CF. The number of death cells was compared in hearts that were exposed to aerobic conditions and hearts subjected to I/R.

| Determination of protein concentration
Bradford method was used to determine protein concentration in the cardiac tissue and cell homogenates. BSA (heat shock fraction, ≥98%, Sigma-Aldrich) served as the protein standard. For measuring total protein concentration, Bio-Rad Protein Assay Dye Reagent (Bio-Rad) and Spark multimode microplate reader (Tecan Trading AG) were used.

| Statistical analysis
Experimental data were analysed using GraphPad Prism 6 software (GraphPad Software). Shapiro-Wilk normality test or Kolmogorov-Smirnov test was used to assess normality of variances changes.
The Student's t test or Mann-Whitney U test was used for comparison between two groups of measurement data. ANOVA or nonparametric test with post hoc tests for multiple groups was used.
Correlations were assessed using Pearson's or Spearman's test, as appropriate. Results were expressed as mean ± SEM, and a value of P < .05 was regarded as statistically significant.

| Enhanced expression of Klotho in human cardiomyocytes subjected to I/R injury
The analysis showed significantly higher expression of Klotho mRNA in myocytes subjected to I/R in comparison with aerobic control group (Figure 2A). Then, we checked if increased expression of Klotho mRNA was accompanied by increased protein synthesis. As shown in Figure 2B, the level of Klotho protein was significantly higher in cells that underwent I/R. We have also observed significantly higher cell F I G U R E 2 Klotho in human cardiomyocytes subjected to I/R injury. A, An expression of Klotho gene in human cardiomyocytes was examined by RT-qPCR and normalized to G6PD, n = 6-12. B, Quantitative analysis of Klotho protein in human cardiomyocytes by ELISA. Klotho protein concentration was normalized to total protein concentration, n = 18. G6PD, glucose-6-phosphate dehydrogenase; I/R, ischaemia/reperfusion; *P < .05 vs aerobic control; all data are expressed as mean ± SEM surface expression of Klotho protein in the cardiomyocytes subjected to I/R, tested by the immunofluorescence staining ( Figure 3A,B). The number of cells in I/R group was significantly lower in comparison with aerobic control, due to damage and apoptosis ( Figure 3C).

| Decreased mechanical function due to heart injury during I/R
Cardiac mechanical function was decreased in hearts subjected to I/R in comparison with aerobically perfused hearts, showing increased heart injury (Table 1; Figure 4A). The release of LDH from the hearts (marker of cell injury) was significantly higher in I/R group, which showed damage of cardiac cells due to I/R ( Figure 4B). The activity of death cell proteases tested by cytotoxicity assay was significantly higher in coronary effluents from hearts subjected to I/R in comparison with aerobic group, indicating increased number of death cells in hearts injured by I/R ( Figure 4C).

| Klotho protein in the rat hearts during I/R injury
To document the expression of Klotho protein at the tissue level, an isolated rat heart model was used. Immunoblot analysis showed that Klotho protein was expressed in the rat heart tissue as a band with apparent molecular mass at approximately 65 kDa ( Figure 5A). There was a significantly increased tissue expression of Klotho protein and its release to coronary effluents in I/R group in comparison with aerobic group ( Figure 5B,C). The content of Klotho in coronary effluents negatively correlated with cardiac mechanical function (P < .05, r = −.7) and positively correlated with LDH level (P < .05, r = .6) ( Figure 5D,E).

| An influence of Klotho protein on metabolic activity of cardiomyocytes
To explore an impact of Klotho on the metabolic activity of cardiomyocytes, the vital inclusion dye FDA and the vital exclusion dye

| D ISCUSS I ON
As the discovery of Klotho, a significant amount of efforts has been devoted to characterize its protective role in oxidative stress, inflammation, fibrosis and apoptosis. 6,[9][10][11]23 It is well established that Klotho acts prophylactic and therapeutic in acute renal failure or chronic kidney disease. 12,14,24 Therefore, the main aim of this study was to identify the role of Klotho protein in the ischaemic injury of heart. In the present study, the compensatory production and release of Klotho protein from cardiomyocytes after ischaemic damage were shown. Our research confirmed that Klotho protein effectively reduced damage and apoptosis of the cardiomyocytes during I/R, showing cardioprotection. We strongly suggest that Klotho protein can be valuable both as a marker of injury and as a potential cardio-   Our results suggest Klotho involvement in the process of oxidative stress and apoptosis during I/R. Given that, we propose a compensative production of Klotho in the cardiomyocytes under stress-related conditions to protect cardiac tissue against ischaemic damage.
Interestingly, Klotho level was depleted in the pancreatic islets in patients with type 2 diabetes mellitus. 27 The compensatory production of Klotho, thus renoprotection, has not been found also in renal disorders. It was reported that renal I/R injury reduced Klotho levels in the kidney tissue and serum in animal models. [34][35][36] Similarly, the renal transplant recipients who developed delayed graft function (DGF) showed a dramatic reduction in renal and plasma Klotho levels. 35  These findings confirm the beneficial influence of Klotho on cell metabolism and survival observed in our research. Researchers emphasize that recombinant Klotho may be prophylactic and therapeutic in progression of acute to chronic kidney disease, renal fibrosis and uremic cardiomyopathy. 14,37 Moreover, it was reported that Klotho reduced apoptosis in experimental ischaemic acute kidney injury or renal and cerebral I/R injury. 13,24,36 Therefore, we propose that Klotho may be considered as a potential therapeutic agent also in I/R injury of heart in the future.

| SUMMARY
Our research showed that: (a) there is an increased compensative production of Klotho protein in the cardiomyocytes during I/R to protect ischaemic heart from further injury; (b) an enhanced production of Klotho during I/R and its release to extracellular space can be used as a marker of heart damage; and (c) Klotho administration protects the cardiomyocytes against I/R injury and improves their metabolism after ischaemic damage.
In conclusion, this study confirmed the potential cardioprotective role of Klotho in the development of I/R injury. We propose that administration of Klotho protein into hearts may prevent the cardiomyocytes from the damage or reduce the area of injury. We strongly suggest that Klotho protein can be valuable both as a marker of injury and as a potential cardioprotective agent and contributes to compensatory mechanism which mitigates the initial damage under cardiac stress.
We are also aware that further studies on the role of exogenous Klotho on cardioprotection on in vivo model are needed. So the next goal of our study is to check whether administration of Klotho before I/R or during I/R will be cardioprotective, and how this cardioprotection is induced.

ACK N OWLED G EM ENTS
We kindly thank Wrovasc for allowing the Langendorff system apparatus and Bio-Rad for allowing ZOE Fluorescent Cell Imager to be rented.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R S ' CO NTR I B UTI O N S
All authors participated in the design, interpretation of the studies, analysis of the data and review of the manuscript; AO, AKZ and IBL designed the research study; AO, AKZ, MB and IBL performed the research; AO and IBL analysed the data; AO wrote the paper; IBL made critical revision of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.