Recombinant Klotho Protein Ameliorates Myocardial Ischemia/Reperfusion Injury by Attenuating Sterile Inflammation

Abstract

Currently, no effective therapy and potential target have been elucidated for preventing myocardial ischemia and reperfusion injury (I/R). We hypothesized that the administration of recombinant klotho (rKL) protein could attenuate the sterile inflammation in peri-infarct regions by inhibiting the extracellular release of high mobility group box-1 (HMGB1). This hypothesis was examined using a rat coronary artery ligation model. Rats were divided into sham, sham+ rKL, I/R, and I/R+ rKL groups (n = 5/group). Administration of rKL protein reduced infarct volume and attenuated extracellular release of HMGB1 from peri-infarct tissue after myocardial I/R injury. The administration of rKL protein inhibited the expression of pro-inflammatory cytokines in the peri-infarct regions and significantly attenuated apoptosis and production of intracellular reactive oxygen species by myocardial I/R injury. Klotho treatment significantly reduced the increase in the levels of circulating HMGB1 in blood at 4 h after myocardial ischemia. rKL regulated the levels of inflammation-related proteins. This is the first study to suggest that exogenous administration of rKL exerts myocardial protection effects after I/R injury and provides new mechanistic insights into rKL that can provide the theoretical basis for clinical application of new adjunctive modality for critical care of acute myocardial infarction.

Keywords: acute myocardial infarction; high mobility group box-1; klotho; myocardial ischemia/reperfusion injury; sterile inflammation.

Figure 1

Recombinant klotho (rKL) reduces infarct volume after myocardial I/R injury. (A) Experimental schedule; (B) Volume of myocardial infarct area stained with of 2,3,5-triphenyltetrazolium chloride (TTC) at 4 h, *** p < 0.001, comparison of myocardial I/R with rKL, one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (C) Representative image of 2,3,5-triphenyltetrazolium chloride (TTC) staining at 4 h; (D) Volume of myocardial infarct area stained with TTC observed at 24 h, * p < 0.05, comparison of myocardial I/R with rKL, unpaired t-test. (E) Representative image of 2,3,5-triphenyltetrazolium chloride (TTC) staining at 24 h (the number of animals: n = 5, respectively).

Figure 2

Intracellular reactive oxygen species (ROS) level. (A) Representative intracellular reactive oxygen species (ROS) results observed following administration of klotho after myocardial ischemic/reperfusion (I/R) injury; (B) ROS staining area, *** p < 0.001, comparison of myocardial I/R with and without administration of recombinant klotho (rKL) by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (number of animals: n = 5). Scale bar, 50 µm.

Figure 3

Recombinant klotho (rKL) suppresses the extracellular release of high mobility group box-1 (HMGB1) after myocardial ischemic/reperfusion (I/R) injury. (A) Representative immunohistochemistry results for rKL treatment after myocardial I/R injury; (B) Immunohistochemistry results, *** p < 0.001, comparing myocardial I/R with rKL using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (the number of animals: n = 5, respectively). (C) Levels of circulating of HMGB1 in plasma, *** p < 0.001, comparison of myocardial ischemic/reperfusion (I/R) injury with and without recombinant klotho (rKL) administration by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (the number of animals: n = 5, respectively). (D) Levels of cardiac troponin T (cTnT) in plasma, *** p < 0.001, comparison of myocardial ischemic/reperfusion (I/R) injury with and without recombinant klotho (rKL) administration by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (the number of animals: n = 5, respectively). Scale bar, 50 µm.

Figure 4

Cardiac mRNA expression of three major inflammatory cytokines in the peri-infarcted regions. (A) Quantification of the expression of tumor necrosis factor-α (TNF-α) by RT-PCR, * p < 0.05, comparison of myocardial ischemic/reperfusion (I/R) injury treated with klotho by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. (B) Quantification of interleukin-1β (IL-1β) expression by RT-PCR, *** p < 0.001. Comparison of myocardial I/R injury treated with recombinant klotho (rKL) by ANOVA followed by Bonferroni post hoc test. (C) Quantification of IL-6 expression by RT-PCR, * p < 0.05. Comparison of myocardial I/R treated with klotho by ANOVA followed by Bonferroni post hoc test (the number of animals: n = 5, respectively).

Figure 5

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. (A) Representative TUNEL assay results for recombinant klotho (rKL) administration after myocardial ischemic/reperfusion (I/R) injury; (B) TUNEL assay results, *** p < 0.001, comparison of myocardial I/R treated with rKL by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test. Scale bar, 50 µm.

Figure 6

Expression levels of several cytokines. (A) Representative chemiluminescence images of the proteome profiler array after administration of klotho following myocardial I/R injury. The dotted line box indicates the selected candidate cytokines. (B) Expression levels of several cytokines, * p < 0.05, ** p < 0.01, comparison of myocardial ischemic/reperfusion (I/R) injury with and without recombinant klotho (rKL) administration by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test (the number of animals: n = 5, respectively).