CTRP12 alleviates cardiomyocyte ischemia‑reperfusion injury via regulation of KLF15

Abstract

Myocardial ischemia‑reperfusion (I/R) serves a crucial role in myocardial infarction. C1q/TNF‑related protein 12 (CTRP12) is a secretory protein involved in metabolism. It has been reported that CTRP12 participates in the regulation of numerous cardiovascular diseases. However, its role in myocardial I/R injury remains unclear. In the present study, the left anterior descending coronary artery in mice was ligated to establish a mouse I/R model. A myocardial hypoxia‑reoxygenation (H/R) cell model was also established. Cardiomyocyte injury was evaluated using hematoxylin and eosin staining, Cell Counting Kit‑8 and a lactate dehydrogenase (LDH) kit. The expression levels of CTRP12 and Krueppel‑like factor 15 (KLF15) in murine myocardial tissues and H9c2 cells were determined using reverse transcription‑quantitative PCR and western blotting, as KLF15 was previously reported to protect against I/R‑induced cardiomyocyte damage. Furthermore, inflammatory factors TNF‑α, IL‑1β and IL‑6 were analyzed using ELISA while apoptosis was assessed using TUNEL assays and western blotting. Moreover, the activity of the CTRP12 promoter was determined using a dual‑luciferase reporter assay. The results demonstrated that I/R surgery markedly exacerbated myocardial tissue damage, whereas H/R treatment significantly reduced cell viability and significantly increased LDH activity as well as the release of inflammatory factors and apoptosis. I/R and H/R induction significantly reduced the expression levels of CTRP12 and KLF15. CTRP12 overexpression significantly alleviated H/R‑induced cell injury and significantly inhibited inflammation and apoptosis. Further analysis demonstrated that KLF15 could significantly promote the activity of the CTRP12 promoter. However, following CTRP12 knockdown, KLF15 overexpression exacerbated cell injury, inflammation and apoptosis. In conclusion, the present study demonstrated that CTRP12 may mitigate inflammation and apoptosis in H/R‑induced cardiomyocytes, possibly via the regulation of KLF15, which provided a theoretical basis for the potential treatment of I/R‑induced myocardial infarction.

Keywords: C1q/tumor necrosis factor‑related protein 12; Krueppel‑like factor 15; apoptosis; inflammation; myocardial ischemia‑reperfusion injury.

Figure 1.

CTRP12 expression is downregulated in I/R-induced myocardial tissue and H/R-induced cardiomyocytes. (A) Representative images of myocardial tissue in control or I/R-induced mice. The arrows indicate lesions in the tissue. Scale bar, 50 µm. (B) mRNA and (C) protein expression levels of CTRP12 were detected in myocardial tissue in I/R-induced mice via RT-qPCR and western blotting, respectively. (D) mRNA and (E) protein expression levels of CTRP12 were detected in H/R-induced H9c2 cells via RT-qPCR and western blotting respectively. Results are presented as the mean ± SD analyzed using three independent experiments. **P<0.01, ***P<0.001 vs. control. CTRP12, C1q/TNF-related protein 12; I/R, ischemic/reperfused; RT-qPCR, reverse transcription-quantitative PCR, H/R, hypoxic/reoxygenated.

Figure 2.

CTRP12 overexpression protects against H/R-induced cell injury in H9c2 cells. (A) Transfection efficiency of Ov-CTRP12 was assessed via reverse transcription-quantitative PCR. (B) mRNA and (C) protein expression levels of CTRP12 in untreated or H/R-induced H9c2 cells transfected with Ov-CTRP12 were detected. (D) Cell viability was assessed using the Cell Counting Kit-8 assay. (E) LDH activity levels were evaluated. Results are presented as the mean ± SD analyzed using three independent experiments. ***P<0.001 vs. control; and ^#P<0.05, ^###P<0.001 vs. H/R group. CTRP12, C1q/TNF-related protein 12; H/R, hypoxic/reoxygenated; Ov, overexpressed; LDH, lactate dehydrogenase; NC, negative control.

Figure 3.

Overexpression of CTRP12 inhibits the release of inflammatory factors and apoptosis in H/R-induced H9c2 cells. (A) TNF-α, (B) IL-1β and (C) IL-6 levels were quantified using ELISA. (D and E) Cell apoptosis was detected using the TUNEL assay. Scale bar, 100 µm. (F) Western blotting was performed to assess protein expression levels of Bcl-2, Bax, cleaved caspase-3 and cleaved PARP. Results are presented as the mean ± SD analyzed using three independent experiments. ***P<0.001 vs. control; and ^##P<0.01, ^###P<0.001 vs. H/R group. CTRP12, C1q/TNF-related protein 12; H/R, hypoxic/reoxygenated; PARP, poly(ADP-ribose) polymerase; Ov, overexpressed; NC, negative control.

Figure 4.

KLF15 regulates CTRP12 expression levels in H9c2 cells. (A) mRNA and (B) protein expression levels of KLF15 were determined via RT-qPCR and western blotting, respectively, in myocardial tissue in I/R-induced mice. (C) mRNA and (D) protein expression levels of KLF15 were determined using RT-qPCR and western blotting, respectively, in H/R-induced H9c2 cells following exposure to H/R compared with the control. (E) Transfection efficiency of Ov-KLF15 was assessed using reverse transcription-quantitative PCR. (F) mRNA and (G) protein expression levels of KLF15 in H/R cells were detected following transfection with Ov-KLF15. (H) mRNA and (I) protein expression levels of CTRP12 were assessed following transfection with Ov-KLF15. (J) Binding site of KLF15 and CTRP12 promoter was predicted using the JASPAR database. (K) CTRP12 promoter activity was determined using the dual-luciferase reporter assay. Results are presented as the mean ± SD analyzed using three independent experiments. **P<0.01, ***P<0.001 vs. control; and ^###P<0.001 vs. H/R group. KLF15, Krueppel-like factor 15; CTRP12, C1q/TNF-related protein 12; I/R, ischemic/reperfused; H/R, hypoxic/reoxygenated; RT-qPCR, reverse transcription-quantitative PCR; Ov, overexpressed; NC, negative control; TSS, transcriptional start site.

Figure 5.

Overexpression of KLF15 protects H/R induced H9c2 cell injury via regulation of CTRP12. (A) Transfection efficiency of Ov-CTRP12 was assessed using reverse transcription-quantitative PCR. (B) Cell viability was assessed using the Cell Counting Kit-8 assay. (C) LDH activity levels were quantified following co-transfection with siRNA-CTRP12 and Ov-KLF15. Results are presented as the mean ± SD analyzed using three independent experiments. ***P<0.001 vs. control; ^##P<0.01, ^###P<0.001 vs. H/R group; and ^∆P<0.05, ^∆∆∆P<0.001 vs. Ov-KLF15 + siRNA-NC + H/R. KLF15, Krueppel-like factor= 15; CTRP12, C1q/TNF-related protein 12; H/R, hypoxic/reoxygenated; LDH, lactate dehydrogenase; siRNA, small interfering RNA; Ov, overexpressed; NC, negative control.

Figure 6.

KLF15 overexpression suppresses H/R induced inflammation and apoptosis in H9c2 cells by regulating CTRP12. (A) TNF-α, (B) IL-1β and (C) IL-6 levels were quantified using ELISA. (D and E) Cell apoptosis was detected using the TUNEL assay. Scale bar, 100 µm. (F) Western blotting was performed to assess protein expression levels of Bcl-2, Bax, cleaved caspase-3 and cleaved PARP. Results are presented as the mean ± SD analyzed using three independent experiments. ***P<0.001 vs. control; ^###P<0.001 vs. H/R group; and ^∆∆P<0.01, ^∆∆∆P<0.001 vs. Ov-KLF15 + siRNA-NC + H/R. KLF15, Krueppel-like factor 15; CTRP12, C1q/TNF-related protein 12; H/R, hypoxic/reoxygenated; PARP, poly (ADP-ribose) polymerase; Ov, overexpressed; siRNA, small interfering RNA; NC, negative control.