Recombinant Klotho Protein Ameliorates Myocardial Ischemia/Reperfusion Injury by Attenuating 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).