The Preventive Effect Of Stromal Cell Derived Factor-1β On High Fat-induced Cardiac Cell Apoptosis And Its Mechanisms

Background:Diabetes can cause damages or even failures on many organs and tissues. With the improvement of living standard, more people suffered obesity, and morbidity of type 2 diabetes increase gradually by year, diabetic cardiomyopathy (DCM), as one of its major complications has been received more attentions than ever before. Lipotoxicity can cause cardiac cell death which plays a critical role in the pathogenesis of DCM, but its exact mechanisms remain largely unknown.The chemokine stromal cell derived factor (SDF-1) regulates many essential biological processes, including cardiac and neuronal development, stem cell mobilization, neovascularization, angiogenesis, tumorigenesis and metastasis. It is generally believed that SDF-1 mediates these many disparate processes via its classic specific cell surface receptor known as chemokine receptor 4 (CXCR4). However, increasing evidences indicate the existence of an alternate receptor, CXCR7, which does not widely express and has different biological functions compared to CXCR4.Previous studies regarding the effect of SDF-1 on cardiac system have mainly been focused on the homing of stem cells into the damaged heart and its role in recovering the damaged heart via its angiogenesis action. However, whether SDF-1 has direct protection of the cardiac cells from lipotoxic effect has not been approached.Objectives:By using palmitate (Pal), as one of saturated free fatty acid, to mimic the environment of hyperlipidemia under the condition of type 2 diabetes, we have several objectives for the present study to be determined: First, to elucidate the possible mechanisms of Pal-induced cardiac cell death. Second, to observe whether SDF-1βcould protect the cardiac cells from Pal-induced apoptosis, and if so, to explore the possible mechanisms. To these ends, in vitro cultured cardiac H9c2 cells, in combination of pharmacological inhibitors and siRNA approaches were used.Methods:1. Pal-induced H9c2 cell death and its mechanisms(1) H9c2 cells were exposed to Pal for dose and time course studies. Cell death ELISA assay and western-blot assay were used to detect DNA fragmentation and cleaved-caspase3 separately.(2) To determine which apoptosis pathway involved in Pal-induced cardiac cell death, we examined the critical proteins of relevant apoptosis pathway (p-p53, p53, Bax, Bcl-2, AIF, GRP78, caspase12, CHOP and caspase8) by western-blot assay.(3) 3-NT accumulation was determined by western-blot assay to observe whether Pal induced nitrosative damage or not.(4) Different inhibitors or scavengers (apocynin, MnTMPyP, L-NAME, urate and 4-PBA) were used to determine 3-NT formation and clarify the relationship of nitrosative damage, ER stress and apoptosis.2. The preventive effect of SDF-1βon Pal-induced cardiac cell death and possible mechanisms(1) Dose effect of SDF-1βon Pal-induced cell death was determined by DNA fragmentation via cell death ELISA assay and cleaved-caspase3 via western-blot assay.(2) Different pharmacological inhibitors or siRNAs (PI3K inhibitor: LY294002; ERK inhibitor: U0126; p38MAPK inhibitor: SB203580; AMPK inhibitor: Compound C; SDF-1/CXCR4 inhibitor: AMD3100; IL-6 siRNA; p38βMAPK siRNA; CXCR7 siRNA) were used to observe whether they can block the protective effect of SDF-1βor not. Furthermore, specific activator (AMPK activator: AICAR) or stimulant (rIL-6) were also used to observe whether they had same protective effect as SDF-1βon Pal-induced apoptosis.Results:1. The best dosage and time point for Pal-induced H9c2 cell apoptosis is Pal at 62.5μM for 15 h.2. The expression of GRP78, CHOP and caspase12 were up-regulated after Pal treatment; Bax/Bcl-2 ratio, p-p53/p53 ratio, AIF and caspase8 were no changes after Pal treatment. These results suggest ER stress pathway, but not mitochondrial pathway or death receptor pathway was involved in Pal-induced cardiac cell death.3. Pal can induce cardiac cell nitrosative damage due to the increase of 3-NT accumulation.4. Pal-induced cardiac cell apoptosis was through nitrosative damage-mediated ER stress production.5. SDF-1βat 100nM can significantly inhibit Pal-induced cell apoptosis.6. The preventive effect of SDF-1βon Pal-induced cardiac cell death was through p38 MAPK activation. P38 inhibitor (SB203580) can totally block the protective effect of SDF-1β, and specific p38βsiRNA had same effect as SB203580.7. The preventive effect of SDF-1βon Pal-induced cardiac cell death was through AMPK activation, and p38 MAPK activation was AMPK dependent. AMPK inhibitor (compound C) can abolish SDF-1βprotective effect, whereas AMPK activator (AICAR) has same protective effect as SDF-1β.8. SDF-1βcan stimulate IL-6 production, and this effect was AMPK and p38 MAPK dependent.9. Specific IL-6 siRNA can abolish protective effect of SDF-1β, whereas recombinant IL-6 has same protective effect as SDF-1β.10. CXCR7 exists on the surface of H9c2 cardiac cells. The preventive effect of SDF-1βon Pal-induced cell death is mediated via CXCR7 but not CXCR4, specific CXCR7 siRNA can totally abolish SDF-1βprotective effect through inhibiting AMPK-mediated p38 MAPK activation. Conclusions:1. Exposure of H9c2 cells to Pal causes nitrosative damage and ER stress related apoptotic cell death.2. SDF-1βcan protect cardiac cells from Pal-induced nitrosative damage, ER stress and cell death.3. The cardiac protective effect of SDF-1βis mediated through its interaction with CXCR7, and activation of AMPK and p38 MAPK-mediated IL-6 production.Innovations:1. We elucidated the mechanisms of Pal-induced H9c2 cardiac cell death, i.e. Pal causes nitrosative damage and ER stress related apoptotic cell death.2. We demonstrated the protective effect of SDF-1βon Pal-induced cardiac cell death.3. We demonstrated that CXCR7 exists on the surface of H9c2 cells.4. We elucidated the mechanisms of SDF-1β's preventive effect on Pal-induced cardiac cell apoptosis through its interaction with CXCR7, and activation of AMPK and p38βMAPK-mediated IL-6 production.