Cathelicidin-related antimicrobial peptide protects against myocardial ischemia/reperfusion injury

Abstract

Background: Cathelicidins are a major group of natural antimicrobial peptides which play essential roles in regulating host defense and immunity. In addition to the antimicrobial and immunomodulatory activities, recent studies have reported the involvement of cathelicidins in cardiovascular diseases by regulating inflammatory response and microvascular dysfunction. However, the role of cathelicidins in myocardial apoptosis upon cardiac ischemia/reperfusion (I/R) injury remains largely unknown.

Methods: CRAMP (cathelicidin-related antimicrobial peptide) levels were measured in the heart and serum from I/R mice and in neonatal mouse cardiomyocytes treated with oxygen glucose deprivation/reperfusion (OGDR). Human serum cathelicidin antimicrobial peptide (LL-37) levels were measured in myocardial infarction (MI) patients. The role of CRAMP in myocardial apoptosis upon I/R injury was investigated in mice injected with the CRAMP peptide and in CRAMP knockout (KO) mice, as well as in OGDR-treated cardiomyocytes.

Results: We observed reduced CRAMP level in both heart and serum samples from I/R mice and in OGDR-treated cardiomyocytes, as well as reduced LL-37 level in MI patients. Knockdown of CRAMP enhanced cardiomyocyte apoptosis, and CRAMP KO mice displayed increased infarct size and myocardial apoptosis. In contrast, the CRAMP peptide reduced cardiomyocyte apoptosis and I/R injury. The CRAMP peptide inhibited cardiomyocyte apoptosis by activation of Akt and ERK1/2 and phosphorylation and nuclear export of FoxO3a. c-Jun was identified as a negative regulator of the CRAMP gene. Moreover, lower level of serum LL-37/neutrophil ratio was associated with readmission and/or death in MI patients during 1-year follow-up.

Conclusions: CRAMP protects against cardiomyocyte apoptosis and cardiac I/R injury via activation of Akt and ERK and phosphorylation and nuclear export of FoxO3a. Increasing LL-37 might be a novel therapy for cardiac ischemic injury.

Keywords: Apoptosis; CRAMP; Cardiomyocyte; Cathelicidin; Ischemia/reperfusion injury; LL-37.

Fig. 1

CRAMP is reduced in cardiac ischemia/reperfusion (I/R) injury and prevents cardiomyocyte apoptosis. a The level of the mCRAMP peptide was measured by ELISA in the infarct, border, and remote zones of mouse I/R hearts compared to a sham group (n = 5). b The level of the mCRAMP peptide was measured by ELISA in the serum from I/R mice compared to a sham group (n = 5). c qRT-PCRs were performed to measure specific genes expressed in isolated neonatal mouse cardiac myocytes (NMCMs) and fibroblasts (NMCFs) (n = 6). d The level of the mCRAMP peptide was measured by ELISA in NMCMs and NMCFs (n = 9). e The level of the mCRAMP peptide was measured by ELISA in NMCMs treated with oxygen glucose deprivation/reperfusion (OGDR) (n = 9). f, g The ratio of apoptosis after rCRAMP stimulation in OGDR-treated neonatal rat cardiomyocytes (NRCMs) as determined by TUNEL staining (f, n = 4) and Western blot (g, n = 6). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. h The level of rCRAMP mRNA in NRCMs after transfection with siRNAs targeting rCRAMP (n = 3). i, j The ratio of apoptosis after transfection with rCRAMP siRNA in OGDR-treated NRCMs as determined by TUNEL staining (i, n = 4) and Western blot (j, n = 6). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. Scale bar = 100 μm (f, i). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

CRAMP reduces cardiomyocyte apoptosis via activating Akt and ERK1/2. a, b Western blot analysis for Akt and ERK1/2 phosphorylation after treatment of neonatal rat cardiomyocytes (NRCMs) with the rCRAMP peptide (a, n = 6) or the siRNA targeting rCRAMP (b, n = 6), in the presence or absence of oxygen glucose deprivation/reperfusion (OGDR) treatment. All membranes were probed, stripped, and then reprobed for determining the phosphorylation levels of Akt and ERK1/2. c–f The apoptosis of OGDR-treated NRCMs after treatment with the Akt inhibitor MK2206 or the MEK inhibitor PD98059 together with the rCRAMP peptide as determined by TUNEL staining (c, e, n = 4) and Western blot (d, f, n = 6). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. Scale bar = 100 μm (c, e). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

CRAMP increases FoxO3a phosphorylation and nuclear export in apoptotic cardiomyocytes. a, b Western blot analysis for FoxO3a phosphorylation after stimulation with the rCRAMP peptide (a, n = 6) or transfection with the siRNA targeting rCRAMP (b, n = 6) in neonatal rat cardiomyocytes (NRCMs) treated with oxygen glucose deprivation/reperfusion (OGDR) or not. All membranes were probed, stripped, and then reprobed for determining the phosphorylation level of FoxO3a. c, d Western blot analysis for FoxO3a subcellular expression in the cytoplasm or nucleus after stimulation with the rCRAMP peptide (c, n = 6) or transfection with the siRNA targeting rCRAMP (d, n = 6) in NRCMs treated with OGDR or not. Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

CRAMP reduces cardiac I/R injury in vivo. a, b Mice were intraperitoneally injected with the mCRAMP peptide, and the infarct size and myocardial apoptosis after I/R injury were analyzed by 2,3,5-triphenyltetrazolium chloride (TTC) staining (a, n = 11) and TUNEL staining (b, n = 4). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. c, d The ratio of Bax/Bcl-2 and Caspase-3 cleavage, in addition to the Akt, ERK1/2, and FoxO3a phosphorylation levels, were analyzed by Western blot after mCRAMP treatment in both the infarct (c) and border (d) zones of mice I/R hearts (n = 3). All membranes were probed, stripped, and then reprobed for determining the phosphorylation levels of Akt, ERK1/2, and FoxO3a. Scale bar = 20 μm (b). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

CRAMP knockout mice display increased cardiac I/R injury. a, b The infarct size and myocardial apoptosis were measured upon I/R injury of CRAMP^−/− mice as determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining (a, n = 10) and TUNEL staining (b, n = 4). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. c, d The ratio of Bax/Bcl and Caspase-3 cleavage, in addition to the Akt, ERK1/2, and FoxO3a phosphorylation levels, were analyzed by Western blot in both the infarct (c) and border (d) zones of CRAMP^−/− mice I/R hearts (n = 3). All membranes were probed, stripped, and then reprobed for determining the phosphorylation levels of Akt, ERK1/2, and FoxO3a. Scale bar = 20 μm (b). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

Identification of potential regulators of CRAMP and their effect in cardiomyocyte apoptosis. a The protein levels of c-Jun, Rela, VDR, and C/EBPα were analyzed by Western blot in neonatal rat cardiomyocytes (NRCMs) treated with oxygen glucose deprivation/reperfusion (OGDR) (n = 3). b The protein levels of c-Jun, Rela, VDR, and C/EBPα were analyzed by Western blot in heart tissues from I/R mice (n = 4). c–f The effects of c-Jun siRNA and Rela siRNA in OGDR-treated NRCMs as determined by TUNEL staining (c, e, n = 4) and Western blot (d, f, n = 6). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. Scale bar = 100 μm (c, e). Data were expressed as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

c-Jun negatively regulates CRAMP in the control of cardiomyocyte apoptosis. a The cardiomyocyte apoptosis was measured by TUNEL staining after transfection with siRNAs targeting Rela, c-Jun, and rCRAMP in neonatal rat cardiomyocytes (NRCMs) treated with oxygen glucose deprivation/reperfusion (OGDR) (n = 4). Immunofluorescent staining for α-actinin was used to label cardiomyocytes. b The cardiomyocyte apoptosis was measured by Western blot (n = 3) after transfection with siRNAs targeting c-Jun and rCRAMP in NRCMs treated with OGDR. c qRT-PCR for rCRAMP mRNA level in NRCMs transfected with c-Jun siRNA (n = 4). Scale bar = 100 μm (a). Data were expressed as mean ± SD. *P < 0.05 vs. OGDR+si-NC group; **P < 0.01 vs. OGDR+si-NC group; ***P < 0.001 vs. OGDR+si-NC group; ^##P < 0.01 vs. OGDR+si-CRAMP group; ^§§P < 0.01 vs. OGDR+si-Jun group

Fig. 8

The serum level of LL-37 is reduced in patients with myocardial infarction. a The serum level of the human cathelicidin LL-37 was measured by ELISA in patients with myocardial infarction (MI, n = 172) and normal controls (n = 160). b The serum level of LL-37 was measured by ELISA in MI patients with cardiovascular readmission and/or death (n = 27) compared to those without readmission and/or death (n = 53) during the 1-year follow-up. c The serum LL-37/neutrophil ratio was determined in MI patients with cardiovascular readmission and/or death (n = 27) compared to those without readmission and/or death (n = 53) during the 1-year follow-up. d The receiver-operator characteristic (ROC) curve was used to assess the sensitivity and specificity of the serum LL-37/neutrophil ratio in prediction of readmission and/or death in MI patients. Data were expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 9

The proposed mechanism for the protective effect of CRAMP against cardiomyocyte apoptosis and myocardial ischemia/reperfusion (I/R) injury. Induced c-Jun and reduced CRAMP were identified in the heart upon I/R injury. CRAMP protects against cardiomyocyte apoptosis and myocardial I/R injury via the activation of Akt and ERK1/2 pathways and the phosphorylation and nuclear export of FoxO3a