C1 inhibitor-mediated myocardial protection from chronic intermittent hypoxia-induced injury

Abstract

The optimal treatment for chronic intermittent hypoxia (CIH)-induced cardiovascular injuries has yet to be determined. The aim of the current study was to explore the potential protective effect and mechanism of a C1 inhibitor in CIH in the myocardium. The present study used a rat model of CIH in which complement regulatory protein, known as C1 inhibitor (C1INH), was administered to the rats in the intervention groups. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The expression of proteins associated with the apoptotic pathway, such as B-cell lymphoma 2 (Bcl-2), Bax and caspase-3 were detected by western blot analysis. The expression of complement C3 protein and RNA were also analyzed. C1INH was observed to improve the cardiac function in rats with CIH. Myocardial myeloperoxidase activity, a marker of neutrophil infiltration, was significantly decreased in the C1INH intervention group compared with the CIH control group, and cardiomyocyte apoptosis was significantly attenuated (P<0.05). Western blotting and reverse transcription-polymerase chain reaction analysis indicated that the protein expression levels of Bcl-2 were decreased and those of Bax were increased in the CIH group compared with the normal control group, but the protein expression levels of Bcl-2 were increased and those of Bax were decreased in the C1INH intervention group, as compared with the CIH group. Furthermore, the CIH-induced expression and synthesis of complement C3 in the myocardium were also reduced in the C1INH intervention group. C1INH, in addition to inhibiting complement activation and inflammation, preserved cardiac function in CIH-mediated myocardial cell injury through an anti-apoptotic mechanism.

Keywords: C1 inhibitor; apoptosis; cardiomyocyte; chronic intermittent hypoxia; complement.

Figure 1.

C1INH prevents myocardial cell apoptosis in rats undergoing CIH. Sectioned myocardial tissues from normal control rats and rats with CIH receiving 0.9% NaCl or 40 U/kg C1INH twice weekly for 4 or 8 weeks were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. (A) Color photomicrographs of heart sections showed positive apoptotic nuclei in myocardial tissues at 4 and 8 weeks with in situ detection of DNA nick ends. Representative images from the (a) normal control, (b) CIH (4 weeks), (c) CIH + C1INH (4 weeks), (d) CIH (8 weeks) and (e) CIH + C1INH (4 weeks) groups are shown. Magnification, ×400. (B) Quantitative analysis of TUNEL positively stained myocardial cells (%) was conducted based on the positive apoptotic nuclei staining in myocardial tissues at 4 and 8 weeks. Results (n=10) are presented as the mean ± standard error of the mean. CIH, chronic intermittent hypoxia; C1INH, C1 inhibitor. *P<0.01, vs. the CIH group.

Figure 2.

Effect of C1INH on Bcl-2, Bax and caspase-3 protein expression in the myocardial area of rats undergoing CIH. (A) Western blot analysis conducted to determine Bcl-2, Bax and caspase-3 expression levels in whole protein extracts from myocardial tissues in normal control rats and rats with CIH receiving 0.9% NaCl or 40 U/kg C1INH twice weekly for 4 or 8 weeks. Normoxic myocardial tissues from the normal control group and β-actin served as internal controls. A representative western blot from five independent experiments is shown. (B) Quantitative analysis of the effect of C1INH on Bax expression in myocardial tissues at 4 weeks and 8 weeks during CIH. (C) Quantitative analysis of the effect of C1INH on Bcl-2 expression in myocardial tissues at 4 weeks and 8 weeks during CIH. (D) Quantitative analysis of the effect of C1INH on caspase-3 expression in myocardial tissues at 4 weeks and 8 weeks during CIH. Results (n=10) are presented as the mean ± standard error of the mean. *P<0.01, vs. the CIH group. Bcl-2, B-cell lymphoma 2; CIH, chronic intermittent hypoxia; C1INH, C1 inhibitor. .

Figure 3.

Effect of C1INH on C3 protein expression in the myocardium of rats undergoing CIH. Western blot analysis of C3 expression levels in whole protein extracts from the myocardial tissues in normal control rats and CIH rats receiving 0.9% NaCl or 40 U/kg C1INH twice weekly for 4 or 8 weeks. Normoxic myocardial tissues from the normal control group and β-actin served as internal controls. (A) Representative western blot from five independent experiments. (B) Quantitative analysis of the effects of C1INH on C3 expression levels in myocardium at 4 weeks and 8 weeks during CIH. Results (n=10) are presented as the mean ± standard error of the mean. *P<0.01, vs. the CIH group. CIH, chronic intermittent hypoxia; C1INH, C1 inhibitor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase..

Figure 4.

Effect of C1INH on C3 mRNA expression in the myocardium of rats undergoing CIH. RNA was isolated from the myocardial tissue of normal control rats and rats with CIH receiving 0.9% NaCl or 40 U/kg C1INH twice weekly for 4 or 8 weeks. C3 mRNA expression was analyzed by reverse transcription polymerase chain reaction. β-actin was the internal control. (A) A representative result is shown. (B) Quantitative analysis of the effects of C1INH on C3 mRNA in the myocardium at 4 weeks and 8 weeks during CIH. Results (n=10) are presented as means ± standard error of the mean. The ratio of the relative density of C3 mRNA in the myocardial tissue to that of the normal control was determined. *P<0.01, vs. the CIH group. CIH, chronic intermittent hypoxia; C1INH, C1 inhibitor.