Giricz Zoltán, Koncsos Gábor, Rajtík Tomáš, Varga Zoltán V, Baranyai Tamás, Csonka Csaba, Szobi Adrián, Adameová Adriana, Gottlieb Roberta A, Ferdinandy Péter
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis University, Nagyvárad tér 4, H-1089, Budapest, Hungary.
Pharmahungary Group, Szeged, Hungary.
Lipids Health Dis. 2017 Mar 23;16(1):60. doi: 10.1186/s12944-017-0455-0.
We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart.
Male Wistar rats were fed either normal chow (NORM; n = 9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n = 9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09 mmol/L vs. 2.89 mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot.
Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments.
This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals.
我们之前已表明，在高胆固醇血症中缺血预处理的效率会降低，且自噬对于心脏保护是必要的。然而，单纯的高胆固醇血症是否会干扰自噬或雷帕霉素靶蛋白（mTOR）信号通路尚不清楚。因此，我们研究了单纯的高胆固醇血症是否会调节大鼠心脏中与自噬相关的信号通路或程序性细胞死亡机制，如凋亡和坏死性凋亡。
将雄性Wistar大鼠分为两组，一组喂食普通饲料（正常组；n = 9），另一组喂食含2%胆固醇和0.25%胆酸的饲料（高胆固醇组；n = 9），持续12周。在喂养期结束时，高胆固醇组大鼠的血浆总胆固醇水平比正常组大鼠升高了41%（4.09 mmol/L对2.89 mmol/L）。处死动物后，取出心脏并简要冲洗。将左心室速冻，通过蛋白质印迹法测定自噬、mTOR信号通路、凋亡和坏死性凋亡的标志物。
与对照组相比，单纯的高胆固醇血症与心脏自噬标志物如LC3-II、Beclin-1、Rubicon和RAB7的表达显著降低有关。核糖体S6的磷酸化是mTOR活性的替代标志物，在高胆固醇组样本中增加。凋亡标志物裂解的半胱天冬酶-3在高胆固醇组心脏中增加，而在不同处理组之间未检测到坏死性凋亡标志物RIP1、RIP3和MLKL的表达差异。
这是首次对高胆固醇血症大鼠心脏中的自噬以及凋亡和坏死性凋亡的程序性细胞死亡通路进行全面分析。我们的数据表明，单纯的高胆固醇血症会抑制基础心脏自噬，自噬的减少可能是mTOR信号通路激活的结果。自噬减少伴随着凋亡增加，而单纯的高胆固醇血症并未调节心脏坏死性凋亡。基础自噬的减少和凋亡的增加可能是高胆固醇血症动物中所报道的心脏保护作用丧失的原因。
