Involvement of MAPK and AMPK signaling pathways in cigarette smoke-induced inflammation in human AC16 cardiomyocytes

INTRODUCTION: Cigarette smoke (CS) is the major risk factor of cardiovascular diseases. There is increasing evidence showing oxidative stress and inflammatory responses may play roles in the pathophysiological process; however, the underlying mechanism on CS-induced inflammation is currently unclear. The aim of this study was to investigate whether mitogen-activated protein kinase (MAPK) and AMP-activated protein kinase (AMPK) signalling pathways are involved in CS-induced inflammatory responses in human AC16 cardiomyocytes in vitro. METHODS: The AC16 cell line was cultured in DMEM/F12 containing 12.5% fetal bovine serum, in a CO2 incubator at 37°C. When cells reached 70% confluence, the medium was replaced with a medium consisting of 1% fetal bovine serum 24 hours before treatment. Cigarette smoke medium (CSM) was prepared by bubbling smoke from two cigarettes into 20 mL serum-free medium, which was regarded as 100%. Cells were pretreated with MAPK pathway inhibitors including U0126 (an ERK1/2 inhibitor), SB203580 (a p38 inhibitor) and SP600125 (a JNK inhibitor) and AMPK inhibitor Compound C for 30 minutes before 2% CSM was added and incubated for an additional 24 hours. Supernatant was collected for determination of interleukin (IL)–8 and IL-6 by enzyme-linked immunosorbent assay. Cell lysates were collected for Western blot analysis. RESULTS: CSM caused dose-dependent elevation of IL-8 and reduction of IL-6 in the supernatant of cardiomyocytes. MAPK inhibitors attenuated CSM-induced elevation of IL-8 release but no effect on CSM induced reduction of IL-6 release. On the other hand, Compound C abolished both CSM-induced IL-8 elevation and IL-6 reduction. Furthermore, CSM increased protein expression of anti-oxidative stress enzyme heme oxygenase 1 (HO-1) and quinone oxidoreductase 1 (NQO1), suggesting the existence of a defensive mechanism in cardiomyocytes. CONCLUSION: These findings suggest that MAPK but not AMPK have a differential role in controlling CSM induced IL-8 and IL-6 release via oxidative stress in human cardiomyoctyes in vitro