Content area
Heart ischemia/reperfusion injury (IRI) significantly contributes to cardiac damage through oxidative and nitrosative stress, where the dysregulation of nitric oxide synthases (NOS) and the subsequent activation of matrix metalloproteinases (MMPs) are observed. Klotho is a multifunctional protein crucial in ageing, metabolism, and ion homeostasis. It has been confirmed that Klotho serves as a critical protective factor across multiple biological systems, with its roles in neuroprotection, cardiovascular health, and renal function being particularly noteworthy. This study aimed to investigate the protective role of Klotho protein against oxidative and nitrosative stress in heart IRI through regulation of the NOS/MMP pathway. The study utilised in vitro human cardiomyocyte culture and ex vivo isolated rat hearts subjected to IRI. Recombinant Klotho protein was administered to evaluate its effects on heart mechanical function, gene and protein expression of NOS isoforms, oxidative and nitrosative stress markers, MMP activity, and lipid metabolism. Administration of Klotho significantly improved heart mechanical and contractile function following ischemia/reperfusion. Klotho normalised the expression and synthesis of endothelial NOS and inducible NOS, resulting in reduced production of reactive nitrogen species and attenuated nitrosative stress. It also limited oxidative damage, reflected by decreased protein oxidation, and restored fatty acid metabolism. Additionally, Klotho regulated MMP-2 and MMP-9 synthesis and activity, thereby protecting heart contractile proteins from degradation. The Klotho protein exhibits significant cardioprotection in IRI by mitigating oxidative and nitrosative stress through the modulation of the NOS/MMP signalling pathway. These findings highlight the therapeutic potential of Klotho in managing ischemic heart conditions and myocardial injury.
Introduction
Heart ischemia/reperfusion injury (IRI) results from the temporary blockage of arterial flow, followed by restored circulation. While ischemia alone causes cellular dysfunction and damage, reperfusion exacerbates the injury by triggering inflammatory and oxidative processes, affecting not only the heart but also remote organs1.
The excessive production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), followed by oxidative/nitrosative stress, is one of the main contributors to IRI1. Moreover, an altered expression of nitric oxide synthases (NOSs) and overproduction of nitric oxide (NO) are observed. While NO functions as a vasodilator in the endothelium and as a radical scavenger, during oxidative stress it reacts with superoxide (O2•−) to form peroxynitrite (ONOO−), a powerful oxidant and nitrating agent that can inflict damage on the myocardium2,3. Heart contractile proteins can be directly modified by ROS, which is accompanied by the activation of kinases and transcription factors involved in cardiac hypertrophy2,3. Activation of matrix metalloproteinases (MMPs) by ROS/RNS during oxidative stress results in the proliferation of cardiac fibroblasts, followed by myocardial remodelling and failure. The essential proteolytic enzymes that degrade extracellular proteins and remodel the extracellular matrix are MMPs, including MMP-2 and MMP-92, 3, 4–5. As a consequence of oxidative/nitrosative stress, activated MMPs promote the proteolysis of heart contractile proteins such as troponin, titin, and myosin light chains (MLCs)3, 4–5.
Klotho is a multifunctional protein crucial in ageing, metabolism, and ion homeostasis. Discovered in 1997, Klotho is primarily expressed in the kidneys, with lower levels found in the brain, parathyroid glands, heart, and other tissues6,7. It exists in two main forms: a transmembrane protein that acts as a co-receptor for fibroblast growth factor 23 (FGF23), and a soluble circulating form. Soluble Klotho is cleaved by metalloproteinase domain-containing protein 10 (ADAM10) and ADAM17, and released into the bloodstream, cerebrospinal fluid, and urine6,7. Its primary functions include the regulation of vitamin D and phosphate metabolism and serving as a vital component in ageing processes and neuroprotection. Research has shown that Klotho levels decline with age and in certain chronic and acute conditions, such as chronic kidney disease, congestive heart failure or acute kidney injury. Conversely, higher levels of Klotho are associated with increased longevity and better overall health6, 7, 8–9. The protein’s diverse effects on multiple physiological systems have made it a subject of intense research, with potential implications for understanding and addressing age-related diseases, neurodegenerative disorders, and metabolic conditions6,7. Importantly, research on cell lines and animal models showed a protective effect of Klotho against oxidative stress and apoptosis10,11. Klotho’s involvement in cardiovascular health highlights its potential as both a biomarker and a therapeutic target for managing cardiovascular disease, heart failure, and possibly myocardial infarction (MI)12, 13–14.
We recently showed that Klotho protein inhibits MMP-dependent degradation of contractile proteins in cardiomyocytes during IRI15. This study aimed to evaluate the effect of Klotho protein on IRI and oxidative stress in terms of the NOS/MMP pathway.
Materials and methods
Human cardiomyocyte culture
The primary Human Cardiac Myocytes (HCM) from ScienCell Research Laboratories (Carlsbad, CA, USA) were cultured as described previously15. HCM underwent in vitro chemical IRI with and without the administration of 1 µg/mL of Recombinant Human Klotho protein (5334-KL-025, R&D Systems, Minneapolis, USA), as described in the experimental protocol previously reported15. The concentration of Klotho protein (1 µg/mL) used was selected based on our previous works15, 16–17 and other published studies18, and was shown to be non-toxic and cytoprotective under both aerobic and IRI conditions, as determined by a cell viability test17.
Experimental animals
Adult male Wistar rats, weighing 200–350 g, were procured from the Mossakowski Medical Research Center at the Polish Academy of Sciences in Warsaw, Poland. The rats were housed in pairs per cage under controlled environmental conditions: temperature (22 ± 2 °C), humidity (55 ± 5%), and a 12-hour light/dark cycle. They had ad libitum access to standard laboratory chow and water. All experimental procedures complied with the Guide of the Polish Ministry of Science and Higher Education for the Care and Use of Experimental Animals. The study was performed following the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.0. The study was approved by the Ethics Committee for Experiments on Animals at the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw, Poland (Resolution 002/2020 of January 15, 2020).
Isolation of rat hearts and perfusion with the Langendorff method
Rats were desensitised with buprenorphine (0.05 mg/kg, i.p.) and anaesthetised with sodium pentobarbital (0.5 mL/kg, i.p.). The depth of anaesthesia was confirmed by the loss of the pedal withdrawal reflex. The animals were then euthanised by thoracotomy and quick excision of the heart. The hearts were immediately immersed in ice-cold Krebs-Henseleit Buffer (118 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L KH2PO4, 1.2 mmol/L MgSO4, 3.0 mmol/L CaCl2, 25 mmol/L NaHCO3, 11 mmol/L glucose, and 0.5 mmol/L EDTA, pH 7.4) to rinse. Within 30 s, the hearts were cannulated via the aorta on a Langendorff system (EMKA Technologies, Paris, France). They were perfused at a constant pressure of 60 mmHg with Krebs-Henseleit Buffer (pH 7.4, 37 °C) and continuously gassed with 5% CO2/95% O2. A latex balloon filled with water was connected to a pressure transducer (EMKA Technologies, Paris, France) and introduced into the left ventricle through the mitral valve. The balloon volume was adjusted to maintain a stable left ventricular end-diastolic pressure of 8–10 mmHg during stabilisation and reperfusion. Hemodynamic parameters, including coronary flow (CF), heart rate (HR), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (PED), and intraventricular pressure (dP/dt), were monitored using an EMKA recording system with IOX2 software version 2.8.0.19 (EMKA Technologies, Paris, France, https://www.emkatech.com/product/iox2-software/). Hearts with CF exceeding 28 mL/min or less than 10 mL/min were excluded from the study.
Global ischemia/reperfusion injury of isolated rat hearts
Rat hearts were randomly assigned to one of three groups: aerobic control (aero group), acute myocardial ischemia/reperfusion injury without Klotho (IRI group), and acute myocardial IRI with Klotho administration (IRI + Klotho group). The experimental protocol for heart IRI is illustrated in Fig. 1. Isolated hearts from the IRI groups underwent 25 min of aerobic stabilisation, followed by 22 min of global no-flow ischemia (induced by cessation of the buffer flow), and then 30 min of reperfusion (under aerobic conditions) with or without Klotho protein administration. Hearts in the aerobic group were perfused aerobically for 77 min. Recombinant rat αKlotho protein (RPH757Ra01, Cloud-Clone Corp., USA) was diluted to a final concentration of 0.5 ng/mL with the Krebs-Henseleit buffer. To minimise protein degradation, recombinant Klotho was aliquoted upon delivery and stored at − 80 °C. A fresh aliquot was thawed and added to the buffer immediately before each experiment. The optimal concentration of Klotho protein was determined experimentally in our previous study19, which showed this dose to be the lowest, effective and non-toxic. Higher concentrations of exogenous Klotho protein were associated with deterioration in cardiac function and were therefore excluded from further experiments (data not shown). Klotho was administered with the perfusion buffer during the last 10 min of aerobic stabilisation and the first 10 min of reperfusion (post-global ischemia) (Fig. 1). To determine cardiac mechanical function, the recovery of rate pressure product (RPP) was expressed as the product of HR and LVDP and evaluated at 25 min of the experiment (the end of aerobic perfusion) and 77 min (the end of reperfusion). After the experimental protocol, isolated hearts were immediately immersed in liquid nitrogen and stored at − 80 °C for further analysis. Additionally, 15 mL of coronary effluent was collected at the start of reperfusion (47 min into the experiment) (Fig. 1), concentrated to a final volume of 1 mL using Amicon Ultra-15 Centrifugal Filter Units with Ultracel-10 membrane (EMD Millipore, USA), aliquoted, and frozen at -80 °C for subsequent biochemical analysis.
Fig. 1 [Images not available. See PDF.]
Experimental protocol of heart IRI. IRI—ischemia/reperfusion injury. Figure created with BioRender (https://biorender.com/).
Gene expression
TRIZOL reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for isolation of the total RNA from human cardiomyocytes and heart tissue according to the manufacturer’s instructions. The concentration and purity of RNA were evaluated with a microvolume ultraviolet (UV) spectrophotometer (NanoDrop Lite, Thermo Scientific). The reverse transcription of the pure RNA samples (1000 ng) with iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA) to prepare cDNA according to the instructions provided was conducted. The analysis of gene expression was assessed with the real-time quantitative PCR (RT-qPCR) and CFX96 Real-Time System (BioRad, Hercules, CA, USA) as previously described15. The expression of glucose-6-phosphate dehydrogenase (G6PD) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was used as an internal reference. The sequences of primers 5′-3′ are listed in Table 1.
Table 1. The sequences of primers used in RT-qPCR. eNOS-endothelial nitric oxide synthase; H – human; iNOS-inducible nitric oxide synthase; MMP-2 – matrix metalloproteinase 2; MMP-9 – matrix metalloproteinase 9; R – rat.
Target | Forward primer (5’ − 3’) | Reverse primer (5’ − 3’) |
|---|---|---|
Klotho H | AGGGTCCTAGGCTGGAATGT | CCTCAGGGACACAGGGTTTA |
iNOS H | CACCTTGGAGTTCACCCAGT | ACCACTCGTACTTGGGATGC |
Klotho R | GCTTGTGGTAACAGTGCCAA | GTCCAGGTGTTAAGACGCCA |
iNOS R | CACCTTGGAGTTCACCCAGT | ACCACTCGTACTTGGGATGC |
eNOS R | TGACCCTCACCGATACAACA | CTGGCCTTCTGCTCATTTTC |
MMP-2 R | AGCAAGTAGACGCTGCCTTT | CAGCACCTTTCTTTGGGCAC |
MMP-9 R | GTTGTGGAAACTCACACGCC | GATCCCCAGAGCGTTACTCG |
Preparation of heart homogenates
Frozen hearts were pulverised using a mortar and pestle in liquid nitrogen. The heart tissue was then mechanically homogenised in ice-cold homogenisation 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 10,000×g at 4 °C for 5 min, and the supernatant was collected and stored at − 80 °C for further biochemical analysis.
ELISA tests
An enzyme-linked immunosorbent assay (ELISA) method was used to assess the level of Klotho protein in human cardiomyocytes (orb397071, Biorbyt Ltd., UK), as well as for the quantitative determination of a disintegrin and metalloprotease 17 (ADAM17) (E3409Ra, BT Laboratory, Shanghai, China), inducible NOS (iNOS) (E0740Ra, BT Laboratory, Shanghai, China), endothelial NOS (eNOS) (E0465Ra, BT Laboratory, Shanghai, China), asymmetric dimethylarginine (ADMA) (CSB-E13039R, Cusabio, Houston, USA), dimethylarginine dimethylaminohydrolase 1 (DDAH1) (E2165Ra, BT Laboratory, Shanghai, China), MMP-2 (MMP200, R&D Systems, Minneapolis, MN, USA), MMP-9 (RMP900, R&D Systems, Minneapolis, MN, USA), tissue inhibitor of metalloproteinases 2 (TIMP-2) (ab213923, Abcam, Cambridge, UK), TIMP-4 (SEA130Ra, USCN Life Science Inc., Wuhan, China) proteins, and free fatty acids (FFA) (E0738RA, BT Laboratory, Shanghai, China) in rat hearts. Klotho protein concentration in cardiac tissue was measured with the Rat Klotho ELISA kit (CSB-E14958R, Cusabio, Houston, USA). It was assumed that it reflects primarily endogenously produced Klotho in the heart tissue, as the exogenous recombinant protein was administered during reperfusion only in the initial 10 min of the protocol, and subsequently cleared during the continued 20 min of perfusion. According to the manufacturer’s instructions, the assays were performed using cell/heart homogenates or coronary effluents, and the results were normalised to total protein concentration (in cell/heart homogenates) or coronary flow (in coronary effluents).
Nitrates/nitrites ((NOx)–) level
The amount of total (NOx)– (oxidative products of endogenous NO) in the heart tissue served as a measure of NO production3,16. A commercially available Nitric Oxide Assay Kit (ab65328, Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions for measurement of (NOx)– level was used. The (NOx)– level was assessed in heart homogenates and normalised to total protein concentration.
Evaluation of protein and lipid oxidation
The Protein Carbonyl Content Assay Kit (MAK094, Sigma-Aldrich, St. Louis, MA, USA) was used to assess the level of oxidative stress by analysing protein oxidation. The carbonyl content was measured by derivatizing protein carbonyl groups with 2,4-dinitrophenylhydrazine (DNPH), resulting in the formation of stable dinitrophenyl (DNP) hydrazone adducts. These adducts can be detected spectrophotometrically at 375 nm, with the absorbance being proportional to the amount of carbonyls present. The protein oxidation was assessed in the heart homogenates and normalised to total protein concentration.
The lipid oxidation was evaluated using the TBARS (TCA Method) Assay Kit (Cayman Chemical, Michigan, USA) according to the manufacturer’s instructions. The MDA-TBA adduct, produced from the reaction between malondialdehyde (MDA) and thiobarbituric acid (TBA) at elevated temperature (90–100 °C) and in acidic conditions, was quantified fluorometrically. The lipid oxidation was assessed in the heart homogenates and normalised to total protein concentration.
Zymography
Gelatine zymography for the analysis of MMP activity was performed using the protocol created in our laboratory as described previously3,15. MMP activity was expressed as the activity (AU) per mg of protein from the heart homogenates.
Total protein concentration
The PierceTM BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the total protein concentration in the heart homogenates. Briefly, the bicinchoninic acid (BCA) protein quantification method involves the reduction of Cu2+ to Cu1+ by the protein in an alkaline environment. The absorbance of the samples was measured at 562 nm. The intensity of the colour was directly proportional to the protein concentration in the sample.
Statistical analysis
The data were analysed using the GraphPad Prism version 9.1.0 (221) (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The group size was calculated based on a priori power analysis with an alpha of 0.05 and a power of 0.80. To assess the normality of variance changes, the Shapiro-Wilk normality test was used, and in all the variables p < 0.05 was considered significant. The equality of group variances with the Brown-Forsythe test was assessed. The comparison of data between groups was made with ANOVA, Welch’s ANOVA, or the nonparametric Kruskal-Wallis test with the post hoc tests (Tukey’s or Dunn’s multiple comparisons tests). The correlation analysis was assessed with Pearson’s or Spearman’s tests. Results were expressed as mean ± SD (normal distribution of data) or median + interquartile range (non-normal distribution of data), with a value of p < 0.05 being regarded as statistically significant.
Results
An influence of Klotho protein on heart hemodynamic parameters
A significantly reduced heart mechanical function (Fig. 2A) and heart rate (Fig. 2C) after ischemia in the IRI group were observed compared to the aerobic control. LVDP recovery percentage was significantly lowered in the IRI group, indicating poor contractile function recovery (Fig. 2F). Coronary flow showed no significant differences in the study groups (Fig. 2D). Administration of Klotho protein significantly improved heart mechanical function (Fig. 2A) and heart rate (Fig. 2C), and improved heart contractile function at the beginning of reperfusion (Fig. 2B). The IRI + Klotho group displayed a significant improvement in the recovery of heart mechanical function (Fig. 2E) and contractile function (Fig. 2F) compared to the IRI group.
Fig. 2 [Images not available. See PDF.]
An effect of global IRI and Klotho protein on heart hemodynamic parameters. (a) Heart mechanical function expressed as RPP, calculated as the product of the heart rate and pressure developed in the left ventricle (intraventricular pressure of left ventricle × heart rate/1000); (b) Heart contractile function based on LVDP; (c) Heart rate at different perfusion time points; (d) Coronary flow; (e) Recovery of heart mechanical function. Percent recovery was calculated as the difference between RPP at 25 and 75 min of perfusion; (f) Recovery of heart contractile function. Percent recovery was calculated as a difference between LVDP at 25 and 75 min of perfusion; bpm—beats per minute; CF—coronary flow; IRI—ischemia/reperfusion injury; LV—left ventricle; LVDP—left ventricle developed pressure; RPP—rate pressure product; bar chart: mean ± SD; box plot: boxes—5–75% percentile, whiskers—min to max + median; naero = 7–8; nIRI = 12–13; nIRI+Klotho = 7–8.
The expression of Klotho
Klotho gene expression remained unchanged in human cardiomyocytes following IRI (Fig. 3A), while the treatment with exogenous Klotho reduced protein synthesis (Fig. 3B). However, in rat hearts subjected to IRI the expression of the Klotho gene was enhanced (Fig. 3C) and the synthesis of the Klotho protein was reduced (Fig. 3D). Perfusion of the heart with exogenous Klotho protein normalised the expression of the Klotho gene to the levels observed in the aero group (Fig. 3C), but did not alter the level of Klotho protein (Fig. 3D). The synthesis of ADAM17 in the heart tissue was decreased in the presence of exogenous Klotho (Fig. 3E).
Fig. 3 [Images not available. See PDF.]
The expression of the Klotho in human cardiomyocytes and rat hearts. (a) The expression of Klotho gene in human cardiomyocytes was examined by RT-qPCR and normalised to G6PD; (b) The synthesis of Klotho protein in human cardiomyocytes tested by ELISA; (c) The expression of Klotho gene in rat hearts was examined by RT-qPCR and normalised to GAPDH; (d) The synthesis of Klotho protein in the hearts tested by ELISA; (e) The level of ADAM17 protein in the hearts; ADAM17—a disintegrin and metalloproteinase domain-containing protein 17; G6PD—glucose-6-phosphate dehydrogenase; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; IRI—ischemia/reperfusion injury; mean ± SD; naero = 6–9; nIRI = 7–12; nIRI+Klotho = 5–7.
An influence of IRI and Klotho on NOS expression and nitrosative stress
The expression of eNOS gene in the hearts was significantly downregulated after IRI (Fig. 4A), as compared to the aerobic controls. At the same time, the release of eNOS protein into extracellular space was higher (Fig. 4B). IRI resulted in downregulated expression of iNOS gene in human cardiomyocytes (Fig. 4C). In contrast, a significant increase of iNOS gene in rat hearts was observed (Fig. 4D). The production of iNOS (Fig. 4E) and DDAH1 (Fig. 4G) was enhanced in rat hearts from the IRI group. Klotho contributed to the normalisation of eNOS gene expression (Fig. 4A) and the reduction of eNOS protein release (Fig. 4B) from the heart tissue. The expression of iNOS gene in human cardiomyocytes was normalised to the levels observed in aerobic control after Klotho supplementation (Fig. 4C). Klotho was also related to the regulation of iNOS gene (Fig. 4D) and protein (Fig. 4E) expression in the hearts subjected to IRI, which was followed by a decrease in NOS inhibitor ADMA (Fig. 4F) and DDAH1 levels (Fig. 4G). As a result of the presence of Klotho in the IRI hearts, the intensity of nitrosative stress was diminished (Fig. 4H).
The level of eNOS protein in coronary effluents was negatively correlated with eNOS gene expression (r= -0.74; p = 0.0003) and positively correlated with (NOx)− level (r = 0.53; p = 0.0196) (Fig. 5A) in the hearts. There was a positive correlation of iNOS protein level in the hearts with iNOS gene expression (r = 0.48; p = 0.0317), (NOx)− level (r = 0.55; p = 0.0125) (Fig. 5B), and with eNOS protein level (r = 0.65; p = 0.0.0025) (Fig. 5C). The level of heart ADMA positively correlated with iNOS protein (r = 0.45; p = 0.0443) and DDAH1 level (r = 0.58; p = 0.0183) (Fig. 5D). There was a positive correlation of (NOx)− level with ADMA (r = 0.49; 0.0334) and DDAH1 levels (r = 0.51; p = 0.0295) (Fig. 5E).
Fig. 4 [Images not available. See PDF.]
An influence of Klotho on nitrosative stress. (a) The expression of the eNOS gene in rat hearts normalised to GAPDH; (b) The level of eNOS protein in the coronary effluents; (c) The expression of the iNOS gene in human cardiomyocytes normalised to G6PD; (d) The expression of the iNOS gene in rat hearts normalised to GAPDH; (e) The level of iNOS protein in the heart homogenates; (f) The level of ADMA, NOS inhibitor, in the heart tissue; (g) The level of DDAH1 in rat hearts, responsible for the degradation of ADMA; (h) The level of total nitrate/nitrite (NOx)− in hearts as an indicator of NO production; ADMA—asymmetric dimethylarginine; DDAH1—dimethylarginine dimethylaminohydrolase 1; eNOS—endothelial nitric oxide synthase; G6PD—glucose-6-phosphate dehydrogenase; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; iNOS—inducible nitric oxide synthase; (NOx)−—nitrate/nitrite; column chart: mean ± SD; box plot: boxes—5–75% percentile, whiskers—min to max + median; naero = 5–10; nIRI = 6–13; nIRI+Klotho = 6–7.
Fig. 5 [Images not available. See PDF.]
(a) Correlation of eNOS protein level with eNOS gene expression and (NOx)− level in rat hearts; (b) Correlation of iNOS protein with iNOS gene expression and (NOx)− level in heart tissue (c) Correlation of heart iNOS with eNOS protein level; (d) Correlation of heart ADMA with iNOS protein and DDAH1 levels; (e) Correlation of (NOx)− level with ADMA and DDAH1 levels in hearts; ADMA—asymmetric dimethylarginine; eNOS—endothelial nitric oxide synthase; DDAH1—dimethylarginine dimethylaminohydrolase 1; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; iNOS—inducible nitric oxide synthase; (NOx)−—nitrate/nitrite.
Oxidative stress and lipid metabolism disturbances as a consequence of IRI
IRI resulted in intensified protein oxidation (Fig. 6A), a decrease in total FFA level (Fig. 6B), and enhanced lipid oxidation (Fig. 6C), as indicators of oxidative stress. Administration of Klotho protein significantly reduced oxidative stress (Fig. 6A) and regulated lipid metabolism (Fig. 6B). There was a moderate correlation of protein oxidation with eNOS (r = 0.49; p = 0.0370) and iNOS (r = 0.48; p = 0.0235) protein levels (Fig. 6D). The content of protein carbonyl in heart tissue also positively correlated with (NOx)− level (r = 0.53; p = 0.0196) and total FFA level (r= -0.55; p = 0.0277) (Fig. 6E). There was a positive correlation of lipid peroxidation with iNOS protein level (r = 0.53; p = 0.0300) and negative correlation with total FFA level (r= -0.66; p = 0.0438) (Fig. 6F).
Fig. 6 [Images not available. See PDF.]
An influence of Klotho on oxidative stress after IRI. (a) The level oxidative stress based on the protein carbonyl content in the heart tissue; (b) Total FFA level calculated as the sum of FFA concentration in the heart homogenates and coronary effluents; (c) Lipid peroxidation based on TBARS level in the heart tissue; (d) Correlation of oxidative stress with eNOS and iNOS protein levels; (e) Correlation of oxidative stress, (NOx)− and total FFA level; (f) Correlation of lipid peroxidation, iNOS protein and total FFA levels; eNOS—endothelial nitric oxide synthase; FFA—free fatty acids; iNOS—inducible nitric oxide synthase; MDA—malondialdehyde; (NOx)−—nitrate/nitrite; TBARS—thiobarbituric acid reactive substances; column chart: mean ± SD; bar chart: median with 25–75% percentile; naero = 4–8; nIRI = 6–10; nIRI+Klotho = 6–7.
An influence of Klotho on the synthesis and activity of MMP-2 and MMP-9 in cardiac tissue
The expression of the MMP-2 gene in the hearts after IRI was significantly downregulated (Fig. 7A), while enhanced production (Fig. 7B) and activity (Fig. 7C) of the MMP-2 protein were observed. MMP-2 activity positively correlated with MMP-2 protein level (r = 0.70; p = 0.0028) (Fig. 7D), (NOx)− level (r = 0.52; p = 0.0287), and with oxidative stress (r = 0.64; p = 0.0057) (Fig. 7E). Perfusion of the hearts with Klotho protein during IRI resulted in regulation of MMP-2 protein synthesis (Fig. 7B) and its activity (Fig. 7C) to the levels observed in aerobic conditions.
The expression of the MMP-9 gene was reduced (Fig. 8A) and the production of the MMP-9 protein was augmented (Fig. 8B) in hearts subjected to IRI compared to the aerobic control group. There was a positive correlation of MMP-9 protein level with iNOS protein (r = 0.63, p = 0.0037) and (NOx)− (r = 0.63; 0.0048) levels (Fig. 8C). IRI increased the synthesis of matrix metalloproteinase inhibitors - TIMP-2 (Fig. 8D) and TIMP-4 (Fig. 8F) in the hearts as well. A positive correlation of TIMP-2 with MMP-2 activity (r = 0.50; p = 0.0331) and MMP-9 protein level (r = 0.62; p = 0.0035) (Fig. 8E) was observed. TIMP-4 level was negatively correlated with ADAM17 level (r= -0.60; p = 0.0088) (Fig. 8G). Klotho contributed to the normalisation of MMP-9 gene expression (Fig. 8A) and MMP-9 protein production (Fig. 8B), and reduced the level of TIMP-2 (Fig. 8D) after IRI.
Fig. 7 [Images not available. See PDF.]
An influence of Klotho on MMP-2 in the rat hearts. (a) The expression of the MMP-2 gene; (b) The level of MMP-2 protein in the heart tissue; (c) The activity of 72 kDa MMP-2 protein evaluated by zymography; (d) Correlation of MMP-2 protein level with its activity; (e) Correlation of MMP-2 activity with (NOx)− level and oxidative stress; AU—arbitrary units; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; MMP-2—matrix metalloproteinase-2; (NOx)−—nitrate/nitrite; column and bar charts: mean ± SD; naero = 5–9; nIRI = 6–9; nIRI+Klotho = 6–7.
Fig. 8 [Images not available. See PDF.]
An influence of Klotho on MMP-9 and TIMPs in the rat hearts. (a) The expression of the MMP-9 gene in the hearts; (b) The level of MMP-9 protein in the heart tissue; (c) Correlation of MMP-9 level with iNOS and (NOx)− levels; (d) The level of TIMP-2 in the heart tissue; (e) Correlation of TIMP-2 level with MMP-2 activity and MMP-9 protein level; (f) The level of TIMP-4 in the heart tissue. (g) Correlation of TIMP-4 and ADAM17 levels; ADAM17—a disintegrin and metalloproteinase domain-containing protein 17; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; iNOS—inducible nitric oxide synthase; MMP-2—matrix metalloproteinase-2; MMP-9—matrix metalloproteinase-9; (NOx)−—nitrate/nitrite; TIMP-2—tissue inhibitor of matrix metalloproteinase 2; TIMP-4—tissue inhibitor of matrix metalloproteinase 4; column and bar charts: mean ± SD; box plot: boxes—5–75% percentile, whiskers—min to max + median; naero = 5–10; nIRI = 6–11; nIRI+Klotho = 6–7.
Discussion
Oxidative stress, pH and calcium imbalances, inflammation, metabolic disturbances, autophagy, and apoptosis are key contributors to myocardial IRI20. Various studies on myocardial injury have shown that IRI is associated with ion accumulation, mitochondrial membrane damage, and endothelial dysfunction20. However, the precise molecular mechanisms underlying myocardial IRI are not yet fully understood. To achieve optimal cardioprotection, it is necessary to explore additional or synergistic multi-target treatments and identify new potential therapeutic factors. It has been confirmed that Klotho serves as a critical protective factor across multiple biological systems, with its roles in neuroprotection, cardiovascular health, and renal function being particularly noteworthy21. Ongoing research continues to explore its therapeutic potential, aiming to leverage Klotho’s protective mechanisms in treating age-related diseases and improving health outcomes. The present study demonstrated that exogenous Klotho protein contributed to a reduction of oxidative stress and damage in the hearts subjected to IRI. This effect may be associated with the regulation of the NOS/MMP pathway.
In this study, subjecting isolated rat hearts to the IRI procedure led to a decline in hemodynamic parameters. Administration of Klotho protein supported the recovery of heart mechanical and contractile function, expressed by improved RPP, LVDP and HR, indicating a protective effect. Our results correspond to the research of Wang et al. (2022 and 2023), where the treatment with exogenous Klotho protein improved cardiac function in an in vivo mouse model of MI22,23. Klotho gene overexpression also regulated the cardiac function parameters, including HR and LVDP, in rats subjected to experimental MI24. Additionally, Klotho supplementation mitigated heart diastolic dysfunction in aged mice25 and reduced infarct volume in a rat model of myocardial IRI26. Finally, the high-intensity interval training (HIIT)-induced increase in Klotho plasma and myocardial levels provided cardioprotection and alleviated heart damage following myocardial IRI in rats27. The next stage of this study focused on analysing Klotho expression, where the gene and protein expressions were confirmed in human cardiomyocytes. No significant changes in gene and protein expressions were observed in response to IRI, while incubation of HCM with exogenous Klotho reduced de novo protein synthesis in cells. However, in heart tissue, the analysis revealed upregulated Klotho gene expression after IRI, which was normalised in the IRI + Klotho group. IRI caused a significant reduction in Klotho protein synthesis in the heart tissue as well. The investigation on Klotho indicates different regulatory mechanisms of Klotho expression in the in vitro and ex vivo models in this study, which requires further research. Importantly, the findings indicate some regulatory mechanisms controlling the Klotho expression differences between cell lines and tissues, suggesting that network changes, in addition to transcriptional levels, should be considered28,29. In this study, a decrease in the level of ADAM17 protein was also observed after Klotho administration. ADAM17 is one of the sheddases responsible for cleaving Klotho from the cell membrane, which results in the release of soluble Klotho into circulation6. The presence of soluble Klotho can influence ADAM17 levels. For instance, higher levels of Klotho may lead to decreased ADAM17 synthesis, thereby reducing further shedding of Klotho itself. The balance between Klotho and ADAM17 is critical for maintaining cardiovascular health. Thus, administration of exogenous Klotho may reduce the levels of ADAM17 through feedback interactions that maintain homeostasis in cardiovascular function6,7,30. This effect could be observed in this study; however, it should be considered as hypothesis-generated, as no ADAM17 mRNA expression or enzymatic activity was assessed. Our findings on the heart model align with those of Ramez et al. (2020), where heart IRI lowered the myocardial and plasma levels of Klotho in rats27, and of Xu et al. (2021), who showed that experimental MI attenuated Klotho protein expression in rat myocardial tissue24. The level of Klotho protein in the heart was also reduced after myocardial IRI in diabetic rats, and after a high glucose aggravated hypoxia/reoxygenation injury in cultured primary cardiomyocytes and H9c2 cells31. Conversely, Klotho deficiency was related to impaired heart function and cardiac hypertrophy in ageing mice32, and contributed to heart failure33. The demonstrated role of Klotho protein in the development of cardiovascular diseases makes it a potential candidate for use as a diagnostic biomarker and therapeutic agent in the treatment of vascular diseases34. There is evidence that Klotho protein supplementation protects against the consequences of acute MI, preventing adverse myocardial remodelling and reducing the incidence of heart rhythm disorders35. In one study, through a meta-analysis, it was found that reduced serum Klotho protein levels are an important predictor of increased risk of all-cause mortality, cardiovascular mortality, and progression to end-stage renal failure among patients with chronic kidney disease. However, given the various limitations of the meta-analysis providing evidence for the protective effects of Klotho protein on the cardiovascular and renal systems, there is a clear need for further clinical studies on its potential therapeutic applications36. In the study by Lázaro et al. (2024), it was found that following cardiac rehabilitation after acute cardiac syndrome, plasma concentrations of Klotho protein increase without significant changes in other components of mineral metabolism. Further studies in this area should clarify whether this effect is associated with clinical benefits during cardiac rehabilitation37. The results of many studies to date confirm the importance of the Klotho protein as a new biomarker of acute left ventricular injury immediately after MI. They also point to the Klotho protein as a new component of diagnostic and therapeutic strategies aimed at preventing cardiac events and their consequences in the course of ischemic heart disease. Collectively, these findings highlight Klotho’s crucial role in maintaining proper heart function, and its adequate expression in the heart or corresponding levels in the organism may have protective effects during MI, potentially preventing the development of IRI or mitigating its consequences.
The discrepancy in Klotho gene and protein levels in our study should be discussed. It could be explained by some regulatory mechanisms like post-transcriptional regulation, translation efficiency, or protein degradation38,39. It was revealed that alterations in transcription are linked to translational changes that have opposing effects on the resulting protein levels38. Scientists emphasise that high mRNA does not guarantee high protein expression, due to regulatory complexities in gene expression and protein synthesis38,39. For example, it is not anticipated that induced transcription will immediately result in higher protein levels during stress conditions, as mRNA maturation, export, and translation processes require time. Therefore, there is a delay between the induction of transcription and the subsequent increase in protein levels40. It was also reported that under anaerobic conditions, RNA-binding proteins (RBPs) can bind to target mRNAs and repress their translation as part of the cellular adaptation to hypoxia41. Alternatively, evidence supports a negative feedback mechanism, where the presence/accumulation of a protein, both intracellularly and extracellularly, can lead to decreased transcription of its encoding mRNA42. This could also explain why Klotho administration did not substantially alter the protein synthesis in our study, but more likely influenced the expression of the Klotho gene after IRI in the heart tissue. The half-life of Klotho mRNA remains poorly defined in cardiac tissue, with published values ranging widely across models. Alternative Klotho transcripts containing premature termination codons (PTCs) have also been shown to be degraded by nonsense-mediated mRNA decay (NMD), further supporting the idea of dynamic regulation and rapid mRNA turnover under certain conditions43. Direct evaluation of mRNA stability and Klotho protein degradation dynamics may help clarify the regulatory mechanisms involved. Notably, differences in Klotho gene and protein expression were observed between the in vitro HCM model and the ex vivo perfused rat hearts. These discrepancies may stem from species-specific regulatory factors as well as the inherent biological complexity of whole cardiac tissue, which includes interactions among multiple cell types absent in isolated cardiomyocyte cultures28,38,40. Such variability underscores the importance of employing both models to gain a comprehensive understanding of Klotho’s effects. While this may present challenges for direct translatability, the consistent cardioprotective outcome observed across both systems supports the therapeutic potential of Klotho in the setting of IRI. Additionally, the unchanged ADAM17 levels in the IRI group, despite the lower levels of Klotho protein, suggest that IRI did not affect ADAM17 expression directly and hence, ADAM17 did not contribute to Klotho level regulation under stress conditions. This stability could suggest that other compensatory mechanisms or pathways are at play that maintain ADAM17 levels despite the stress of IRI. The decrease in ADAM17 synthesis after Klotho may indicate some feedback inhibition or protective mechanisms activated by Klotho. However, additional experiments that could clarify these mechanisms in future studies are needed.
It has been well established that overproduction of ROS and RNS, along with the resulting oxidative/nitrosative stress during IRI occurs1. The formation of NO from L-arginine and its bioactivity is regulated by NOS isoenzymes like eNOS, iNOS, neuronal NOS, as well as by endogenous NOS inhibitor - ADMA, and DDAH, a key enzyme that metabolises ADMA. This process is oxygen-dependent, with synthesis rates being proportional to the oxygen concentration3,44. It is known that NOS’s expression changes lead to disturbed NO production, while iNOS can produce large amounts of cytotoxic NO3,44. While NO typically acts as a vasodilator in the endothelium and serves as a scavenger of free radicals, during oxidative stress, it can react with O2•− to form ONOO−. ONOO− is a highly reactive oxidant and nitrating agent that can cause significant damage to myocardial tissue2,3. The present study showed an increased level of eNOS protein in hearts subjected to IRI, whilst the expression of the eNOS gene was lower. The lowered expression of the iNOS gene in HCM was also observed, while the iNOS gene and protein expression were enhanced in rat hearts. This diversity between the NOS gene and protein expression can be explained by the fact that eNOS is constitutively expressed and regulated by transcriptional, post-transcriptional, and post-translational modifications, whereas iNOS is induced primarily by gene transcription44. It is known that cells can regulate the rate of protein synthesis by altering translation efficiency. For instance, specific regulatory elements within the mRNA or interactions with non-coding RNAs, such as microRNAs, can either promote or inhibit translation, thereby compensating for fluctuations in mRNA levels. The negative feedback mechanisms, previously discussed, can also be taken into consideration40. Our results also highlight that regulatory mechanisms in tissue may not be faithfully mimicked by cell models. Cells can exhibit significant gene expression and transcriptional differences compared to tissues28,29,45. Further studies are warranted to investigate these discrepancies using more advanced models, such as 3D cardiac co-cultures, organoids, or single-cell transcriptomics. In this study, eNOS and iNOS levels positively correlated with (NOx)−, suggesting a possible relationship of NOSs with the overproduction of NO and its oxidative products that follow. We also observed a positive correlation of ADMA with iNOS, (NOx)− and DDAH1. The data indicate co-occurrence, which may reflect a possible pathway where nitrosative stress induced ADMA expression, followed by adequate enhancement in DDAH1, which degraded ADMA during IRI. This could be why there was no difference in ADMA level between the aero and IRI groups, but further mechanistic validation is needed to define causality. Our observations agree with the general thesis of the detrimental role of NOSs during heart IRI or MI44. It was proven that during IRI, eNOS-derived NO promotes vasodilation, improving blood flow to ischemic tissues and reducing myocardial injury. eNOS activation was associated with better cardiac function and reduced infarct size post-MI44,46. On the other hand, the induced synthesis of NO by eNOS during IRI contributes to the ONOO− formation and increases oxidative stress within cells, which has important pathological implications44. Thus, the higher expression of NOS proteins in the IRI group may be a compensatory mechanism to enhance NO production during reperfusion in our study. ROS production and oxidative stress can be enhanced by NOS “uncoupling”, where NOS produces O2•− instead of NO. This phenomenon can be observed in eNOS and iNOS, and can be induced by ADMA3,44. The NOS uncoupling might paradoxically result in increased (NOx)− levels due to the reaction between O2•− and NO, forming ONOO−, which can further decompose into nitrate and nitrite. It was also reported that combined exercise training and eNOS overexpression had detrimental effects after MI in mice due to increased oxidative stress from eNOS uncoupling47. The expression of eNOS was elevated in the mouse cardiac tissue after MI48. Increased iNOS activity and subsequent NO and ROS production have also been associated with diabetic cardiomyopathy, endothelial dysfunction and failing heart44. Importantly, here we reported that supplementation of the hearts with Klotho protein contributed to the regulation of eNOS and iNOS gene and protein expression, which resulted in reduced (NOx)− synthesis and protection against nitrosative stress. The decrease in NOS levels in the IRI + Klotho group led to a reduction in ADMA production, because (NOx)− levels declined, eliminating the need for competitive inhibition of NOS. This was accompanied by a decrease in DDAH level. It shows that Klotho contributed to the regulation of the NOS/NO pathway.
Oxidative stress leads to cell damage and death, resulting from the cumulative effects of DNA, protein, and lipid peroxidation2. This study observed significant protein oxidation in IRI hearts as an oxidative stress marker. The positive correlation of protein oxidation with eNOS, iNOS and (NOx)− levels may suggest a potential link between NOS activity and oxidative/nitrosative stress during IRI. However, these correlations are associative and do not establish a causal relationship, which requires further analysis. Then, IRI increased the utility of free fatty acids in the heart, while generating large amounts of lipid peroxidation products. The level of FFA negatively correlated with protein and lipid oxidation, and positively correlated with iNOS level, which may indicate a possible alteration in fatty acid metabolism during oxidative/nitrosative stress. It was established that IRI alters the metabolism of fatty acids, which are the most significant energy suppliers49. Myocardial ischemia leads to the inhibition of fatty acid oxidation, resulting in its accumulation and aggravation of heart injury. However, restoring oxygen supply during reperfusion can cause a temporary overshoot in oxidative metabolism, leading to an increased rate of fatty acid oxidation. Increased fatty acid uptake and metabolism can further deplete fatty acid levels, as the tissues rapidly attempt to restore energy balance. Importantly, abnormal metabolism of fatty acids during IRI stimulates ROS production in cardiac mitochondria, exacerbating cardiac injury as well49,50. The consequence of oxidative stress is also lipid peroxidation, where ROS damage free fatty acids and other lipids50. This oxidative damage can reduce the available pool of functional FFAs, contributing to the lower levels observed in the IRI group. What is important in this study, perfusion of the hearts with Klotho protein reduced oxidative stress and regulated the metabolism of fatty acids to the levels observed in aerobic conditions. It was reported that restoration of higher FFA levels was related to the preservation of systolic function in diabetic rats after MI51. Then, Klotho protected the podocytes against oxidative stress in palmitate-induced injury52. In men after sprint interval training, the level of Klotho in the blood was positively correlated with FFA level53. It should be noted that Klotho is strongly related to oxidative stress defence. Our previous studies demonstrated that Klotho regulated redox balance and supported metabolic homeostasis of human cardiomyocytes15. Then, Klotho prevented oxidative stress after rat heart IRI via insulin-like growth factor receptor (IGF1R)/phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signalling pathway54. In 2023, Wang et al. reported that Klotho improved cardiac fibrosis, inflammation, ferroptosis, and oxidative stress by regulation of AMP-activated protein kinase (AMPK)/mammalian target-of-rapamycin (mTOR) signalling in mice with MI in vivo23. Administration of exogenous Klotho protein reduced intracellular levels of ROS and sterile inflammation in peri-infarct regions26, and reduced oxidative stress via SIRT1 signalling after myocardial IRI in rats as well31. Interestingly, HIIT-induced increase in plasma and myocardial levels of Klotho27 or Klotho gene overexpression24 was related to enhancement in antioxidant production, thus reducing oxidative stress in rats after IRI. Based on these previous findings, Klotho could modulate NOS and MMP activity through the regulation of redox-sensitive signalling pathways such as IGF1R/PI3K/AKT, AMPK/mTOR, or SIRT1, which requires future mechanistic studies. In summary, this study highlights the importance of Klotho’s cardioprotective ability against oxidative stress.
ROS can directly alter heart contractile proteins, which are associated with apoptosis and the activation of kinases and transcription factors involved in cardiac hypertrophy2,3. ONOO− can be formed from (NOx)− and can lead to protein carbonylation and MMP activation, along with other ROS/RNS3,15. MMPs, including MMP-2 and MMP-9, are key proteolytic enzymes that degrade extracellular matrix proteins, are involved in extracellular matrix remodelling, and can be inhibited by TIMP2, 3, 4–5,15. During oxidative and nitrosative stress, the phosphorylation, nitration, and nitrosylation levels of myocardial contractile proteins by (NOx)− and ROS increase. These modified proteins are subsequently degraded by MMPs, resulting in impaired cardiac contractility. The contractile dysfunctions observed during IRI are linked to the degradation of troponin, titin, and myosin light chains (MLCs) by MMPs3, 4, 5–6,15. This study reported significantly increased levels of MMP-2 and MMP-9 proteins in hearts injured by ischemia/reperfusion. The activity of MMP-2 was also enhanced, and positively correlated with MMP-2 protein level, (NOx)− level and protein oxidation. The positive correlation of MMP-9 protein with iNOS and (NOx)− levels was also reported. These findings suggest a possible relationship between oxidative and nitrosative stress with MMP synthesis and activation. Then, we found an intensified synthesis of MMP inhibitors, TIMP-2 and TIMP-4 during IRI, and a correlation of TIMP-2 with MMP-2 and MMP-9, which may reflect a cell reaction to high levels of MMPs. However, the observed associations should be interpreted as indicative of concurrent alterations rather than direct regulatory effects. Aligning with the thesis that TIMP-4 interacts with and inhibits ADAM176,55, there was a negative correlation between TIMP-4 and ADAM17 levels. However, the effectiveness of TIMP-4 is limited compared to stronger inhibitors like TIMP-355. Importantly, we report that Klotho contributed to the limitation of MMP-2 and MMP-9 synthesis/activity, and regulated MMP-2 and MMP-9 gene expression. As a result of MMPs regulation, the level of TIMP-2 was also reduced in the IRI + Klotho group. These results are consistent with our previous report on IRI of human cardiomyocytes, where Klotho supplementation decreased the levels of MMP-2 and MMP-9, thus protecting against MMP-dependent degradation of contractile proteins like MLC1 and troponin I15. Next, we showed that Klotho decreased tissue injury, enhanced antioxidant capacity, and reduced degradation of contractile proteins, supporting the heart’s function in the rat heart IRI ex vivo model19. Interestingly, the levels of MMP-2 and MMP-9 were higher in human cardiac myocytes with Klotho deficiency and subjected to the fibrotic environment, which was mitigated during Klotho overexpression in these cells56. Then, reduced levels of Klotho were related to increased expression of MMP-2 and MMP-9, and atherosclerosis in the primary human aortic smooth muscle cells and diabetic mice57. Wu et al. (2023) revealed that the absence of Klotho in the mouse lacrimal gland resulted in oxidative stress, followed by an increase in MMP-2 and MMP-9 expression, extracellular matrix remodelling, fibrosis, and cell atrophy58. Klotho-deficient mice also showed an increase in MMP-8, MMP-9, and MMP-12 levels in the lungs59. Contrary, the inhibition of MMP and extracellular matrix degradation by Klotho was reported in nucleus pulposus cells60. Klotho gene overexpression decreased the levels of aortic MMP-2 and MMP-9 related to systolic hypertension and arterial stiffness in senescence-accelerated and old mice61. All of the above emphasise Klotho’s contribution to protection related to the inhibition of MMPs, and as we showed, to cardioprotection during IRI as well. Unlike our previous study, which was limited to an in vitro human cardiomyocyte model and focused primarily on MMP-mediated contractile protein degradation, the present work expands the investigation to an ex vivo isolated rat heart model, providing an initial analysis of the NOS/(NOx)−/MMP signalling axis and the effect of Klotho on oxidative/nitrosative stress and extracellular matrix regulation at the tissue level.
Summary
To our knowledge, this is the first study demonstrating that Klotho protein protects against oxidative stress via regulation of the NOS/MMP pathway in the heart IRI ex vivo model.
The highlights of this study are as follows:
Klotho improves heart function and recovery after IRI.
Klotho regulates NOS activity, reducing oxidative and nitrosative stress in heart tissue.
Klotho normalises MMP-2 and MMP-9 levels, protecting heart tissue from degradation.
The findings we have presented are part of the basic sciences embracing the most crucial problems of modern cardiology, which include the consequences of ischemia and reperfusion in the myocardium. Further research on the mechanisms of myocardial ischemia and reperfusion and also on oxidative stress is particularly important, as it allows to understand the essence of these processes. Thus, it creates the basis and possibilities for the development of effective therapeutic methods, particularly in the context of myocardial infarction and heart failure, where oxidative and nitrosative stress play critical roles.
Limitations
We recognise that our study indicates a potential trend, but the absence of detailed mechanistic insights prevents a precise understanding of this phenomenon. Notably, this research represents an initial exploration of the NOS/MMP pathway associated with oxidative stress and heart injury, laying the groundwork for more targeted and detailed analyses in future studies, including inhibitors, activators, or genetic models. These limitations underscore the need for further research to uncover the specific molecular mechanisms through which Klotho influences NOS and MMP activity. Thus, the next step should be the evaluation of Klotho’s effect on NOS/MMP signalling using an in vivo animal model. The exogenous Klotho protein concentrations used in the in vivo HCM culture and ex vivo rat heart perfusion differ due to model-specific requirements. The selected doses represent the most effective non-toxic concentrations validated in prior studies, and direct comparison across models should be interpreted with caution. It also remains unclear whether Klotho acts as a cardioprotective agent, a therapeutic intervention, or both. Consequently, this study focused on the peri-infarction period, administering Klotho protein shortly before and after global ischemia. More precisely, in the ex vivo studies, Klotho was administered during the late stabilisation and early reperfusion phases to mimic the acute clinical setting and to assess its potential both as a preventive and early therapeutic agent. This approach aimed to evaluate whether early Klotho delivery could modulate the pathway before irreversible myocardial injury occurs. Although alternative timings were not explored in the current study due to model limitations, our findings provide a foundation for future in vivo studies to investigate the optimal therapeutic window for Klotho administration. While our results highlight the therapeutic potential of Klotho in myocardial IRI, its clinical translation faces several challenges. Recombinant Klotho has a short half-life, limited stability in circulation, and is rapidly cleared, which complicates its use in acute conditions like MI. Optimal dosing, timing, and delivery route remain undefined, and no clinically approved formulation currently exists. Future studies should explore improved delivery systems or alternative approaches, such as Klotho-mimetics or inducers of endogenous expression, to overcome these limitations.
Author contributions
Conceptualisation: AO; Methodology: AO; Animal Handling: IS; Formal Analysis: AO and IB-L; Investigation: AO; AK-Z, IB-L; Resources: AO and IB-L; Data Curation: AO; Writing – Original Draft Preparation: AO; Writing – Review & Editing: IB-L and AM; Supervision: IB-L; Project Administration: AO; Funding Acquisition: AO.
Funding
This study was funded by the National Science Centre Poland (grant number 2019/33/N/NZ3/01649).
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Competing interests
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
1. Kalogeris, T. et al. Chapter Six—Cell biology of ischemia/reperfusion injury (eds Jeon, K. W.) 229–317. https://www.sciencedirect.com/science/article/pii/B9780123943095000067 (Academic Press, 2012).
2. Tsutsui, H; Kinugawa, S; Matsushima, S. Oxidative stress and heart failure. Am. J. Physiol-Heart Circ. Physiol.; 2011; 301,
3. Krzywonos-Zawadzka, A et al. Cardioprotective effect of MMP-2-inhibitor-NO-donor hybrid against ischaemia/reperfusion injury. J. Cell. Mol. Med.; 2019; 23,
4. Wang, W; Sawicki, G; Schulz, R. Peroxynitrite-induced myocardial injury is mediated through matrix metalloproteinase-2. Cardiovasc. Res.; 2002; 53,
5. Kandasamy, AD et al. Matrix metalloproteinase-2 and myocardial oxidative stress injury: beyond the matrix. Cardiovasc. Res.; 2010; 85,
6. Kanbay, M et al. Klotho: a potential therapeutic target in aging and neurodegeneration beyond chronic kidney disease—a comprehensive review from the ERA CKD-MBD working group. Clin. Kidney J.; 2023; 17,
7. Paquette, J-S et al. The longevity protein klotho: A promising tool to monitor lifestyle improvements. Metabolites; 2023; 13,
8. Hu, M-C et al. Klotho deficiency is an early biomarker of renal ischemia–reperfusion injury and its replacement is protective. Kidney Int.; 2010; 78,
9. Xu, J.-P. et al. Associations between serum soluble α-Klotho and the prevalence of specific cardiovascular disease. Front. Cardiovasc. Med. (2022).
10. Yamamoto, M et al. Regulation of oxidative stress by the anti-aging hormone Klotho. J. Biol. Chem.; 2005; 280,
11. Sugiura, H et al. Klotho reduces apoptosis in experimental ischaemic acute renal failure. Nephrol. Dial Transpl.; 2005; 20,
12. Taneike, M et al. Alpha-Klotho is a novel predictor of treatment responsiveness in patients with heart failure. Sci. Rep.; 2021; 11,
13. Olejnik, A. et al. The biological role of klotho protein in the development of cardiovascular diseases. BioMed Res. Int.https://www.hindawi.com/journals/bmri/2018/5171945/ (2018).
14. Olejnik, A et al. Klotho and FGF23 proteins levels and occurrence of myocardial infarction in patients with acute coronary syndrome. Eur. J. Prev. Cardiol.; 2023; 30,
15. Olejnik, A et al. Klotho protein decreases MMP-Mediated degradation of contractile proteins during ischaemia/reperfusion injury to the cardiomyocytes. Int. J. Mol. Sci.; 2022; 23,
16. Olejnik, A et al. The Klotho protein supports redox balance and metabolic functions of cardiomyocytes during ischemia/reperfusion injury. Cardiol. J.; 2022; 29,
17. Olejnik, A et al. Klotho protein contributes to cardioprotection during ischaemia/reperfusion injury. J. Cell. Mol. Med.; 2020; 24,
18. Lim, SW et al. Klotho enhances FoxO3-mediated manganese superoxide dismutase expression by negatively regulating PI3K/AKT pathway during tacrolimus-induced oxidative stress. Cell. Death Dis.; 2017; 8,
19. Olejnik, A et al. Ameliorating effect of Klotho protein on rat heart during I/R injury. Oxid. Med. Cell. Longev.; 2020; 2020, 6427284. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33123314][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586150]
20. Gunata, M; Parlakpinar, H. A review of myocardial ischaemia/reperfusion injury: pathophysiology, experimental models, biomarkers, genetics and Pharmacological treatment. Cell. Biochem. Funct.; 2021; 39,
21. Prud’homme, G. J., Kurt, M. & Wang, Q. Pathobiology of the Klotho antiaging protein and therapeutic considerations. Front. Aging (2022).
22. Wang, K et al. Cardioprotection of Klotho against myocardial infarction-induced heart failure through inducing autophagy. Mech. Ageing Dev.; 2022; 207, 111714. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35931242]
23. WANG, K et al. Klotho improves cardiac fibrosis, inflammatory cytokines, ferroptosis, and oxidative stress in mice with myocardial infarction. J. Physiol. Biochem.; 2023; 79,
24. Xu, Z et al. Klotho gene improves oxidative stress injury after myocardial infarction. Exp. Ther. Med.; 2021; 21,
25. Daneshgar, N. et al. Klotho inhances diastolic function in aged hearts through Sirt1-mediated pathways. GeroScience. https://doi.org/10.1007/s11357-024-01209-w (2024).
26. Myung, J et al. Recombinant Klotho protein ameliorates myocardial ischemia/reperfusion injury by attenuating sterile inflammation. Biomedicines; 2022; 10,
27. Ramez, M et al. High-intensity interval training increases myocardial levels of Klotho and protects the heart against ischaemia–reperfusion injury. Exp. Physiol.; 2020; 105,
28. Lopes-Ramos, CM et al. Regulatory network changes between cell lines and their tissues of origin. BMC Genom.; 2017; 18,
29. Pontén, F et al. A global view of protein expression in human cells, tissues, and organs. Mol. Syst. Biol.; 2009; 5, 337. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20029370][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824494]
30. Chen, C-D et al. Identification of cleavage sites leading to the shed form of the Anti-Aging protein Klotho. Biochemistry; 2014; 53,
31. Qiu, Z et al. Activation of Klotho/SIRT1 signaling pathway attenuates myocardial ischemia reperfusion injury in diabetic rats. Shock; 2023; [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36533528][DOI: https://dx.doi.org/10.1097/SHK.0000000000002418]
32. Chen, K et al. Klotho deficiency causes heart aging via impairing the Nrf2-GR pathway. Circ. Res.; 2021; 128,
33. Chen, K; Wang, S; Sun, Z. Vivo Cardiac-specific expression of Adenylyl cyclase 4 gene protects against Klotho Deficiency-induced heart failure. Transl Res.; 2022; 244, pp. 101-113. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35114419][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9119924]
34. Martín-Núñez, E et al. Implications of Klotho in vascular health and disease. World J. Cardiol.; 2014; 6,
35. Vazquez, S et al. Cardioprotective effect of the anti-ageing protein Klotho on ischemic heart disease. Cardiovasc. Res.; 2024; 120,
36. Kanbay, M et al. The association between Klotho and kidney and cardiovascular outcomes: a comprehensive systematic review and meta-analysis. Clin. Kidney J.; 2024; 17,
37. Pello Lázaro, AM et al. Cardiac rehabilitation increases plasma Klotho levels. J. Clin. Med.; 2024; 13,
38. Perl, K et al. Reduced changes in protein compared to mRNA levels across non-proliferating tissues. BMC Genom.; 2017; 18,
39. Prabahar, A et al. Unraveling the complex relationship between mRNA and protein abundances: a machine learning-based approach for imputing protein levels from RNA-seq data. NAR Genomics Bioinforma; 2024; 6,
40. Liu, Y; Beyer, A; Aebersold, R. On the dependency of cellular protein levels on mRNA abundance. Cell; 2016; 165,
41. Ho, JJD et al. A network of RNA-binding proteins controls translation efficiency to activate anaerobic metabolism. Nat. Commun.; 2020; 11,
42. Stapleton, JA et al. Feedback control of protein expression in mammalian cells by tunable synthetic translational Inhibition. ACS Synth. Biol.; 2012; 1,
43. Mencke, R. et al. Human alternative Klotho mRNA is a nonsense-mediated mRNA decay target inefficiently spliced in renal disease. JCI Insight2 (20) (2017).
44. Roy, R et al. Dysfunctional and dysregulated nitric oxide synthases in cardiovascular disease: mechanisms and therapeutic potential. Int. J. Mol. Sci.; 2023; 24,
45. Jin, H et al. Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation. Nat. Commun.; 2023; 14,
46. Kumphune, S. et al. Cardiac endothelial ischemia/reperfusion injury-derived protein damage-associated molecular patterns disrupt the integrity of the endothelial barrier. Heliyon10 (2) (2024).
47. de Waard, MC et al. Detrimental effect of combined exercise training and eNOS overexpression on cardiac function after myocardial infarction. Am. J. Physiol-Heart Circ. Physiol.; 2009; 296,
48. Li, J et al. HSPA12B attenuates cardiac dysfunction and remodelling after myocardial infarction through an eNOS-dependent mechanism. Cardiovasc. Res.; 2013; 99,
49. Li, Q et al. Energy metabolism: A critical target of cardiovascular injury. Biomed. Pharmacother; 2023; 165, 115271. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37544284]
50. Tian, H et al. Abnormalities of glucose and lipid metabolism in myocardial ischemia-reperfusion injury. Biomed. Pharmacother; 2023; 163, 114827. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37141734]
51. Malfitano, C et al. Glucose and fatty acid metabolism in infarcted heart from streptozotocin-induced diabetic rats after 2 weeks of tissue remodeling. Cardiovasc. Diabetol.; 2015; 14,
52. Kang, JS et al. Protective effects of Klotho on palmitate-induced podocyte injury in diabetic nephropathy. PLOS ONE; 2021; 16,
53. Middelbeek, RJW et al. Exercise intensity regulates cytokine and Klotho responses in men. Nutr. Diabetes; 2021; 11,
54. Olejnik, A et al. Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury. Sci. Rep.; 2023; 13,
55. Melendez-Zajgla, J et al. Tissue inhibitor of Metalloproteinases-4. The road less traveled. Mol. Cancer; 2008; 7, 85. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19025595][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2599898]
56. Liu, Q et al. The axis of local cardiac endogenous Klotho-TGF-β1-Wnt signaling mediates cardiac fibrosis in human. J. Mol. Cell. Cardiol.; 2019; 136, pp. 113-124. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31520610]
57. Ajay, AK et al. Local vascular Klotho mediates diabetes-induced atherosclerosis via ERK1/2 and PI3-kinase-dependent signaling pathways. Atherosclerosis; 2024; 396, 118531. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38996716]
58. Wu, C-Y et al. Klotho null mutation indirectly leads to Age-Related lacrimal gland degeneration in mutant mice. Biology; 2023; 12,
59. Akasaka-Manya, K et al. Decreased ADAM17 expression in the lungs of α-Klotho reduced mouse. J. Biochem. (Tokyo); 2020; 167,
60. Yi, Y-Y et al. Exogenous Klotho ameliorates extracellular matrix degradation and angiogenesis in intervertebral disc degeneration via Inhibition of the Rac1/PAK1/MMP-2 signaling axis. Mech. Ageing Dev.; 2022; 207, 111715. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35952859]
61. Fan, J et al. Aging impairs arterial compliance via Klotho-mediated downregulation of B-cell population and IgG levels. Cell. Mol. Life Sci.; 2022; 79,
© The Author(s) 2025. This work is published under http://creativecommons.org/licenses/by-nc-nd/4.0/ (the "License"). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.