Hypercholesterolemia downregulates autophagy in the rat heart

Background: 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. Methods: 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 (4.09mmol/L vs. 2.89mmol/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. Results: 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. Conclusions: 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

Hypercholesterolemia downregulates autophagy in the rat heart

Background: 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. Methods: 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 (4.09mmol/L vs. 2.89mmol/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. Results: 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. Conclusions: 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

Determination of Novel Highly Effective Necrostatin Nec-1s in Rat Plasma by High Performance Liquid Chromatography Hyphenated with Quadrupole-Time-of-Flight Mass Spectrometry

Necrostatins have been shown to retard necroptosis, a programmed necrotic-like cell death, which has been shown to underlie pathophysiology of various diseases. Nec-1s, a novel highly effective necrostatin, overcomes some drawbacks of former necrostatin analogues. The determination of Nec-1s in biological system, however, has not been carried out so far. Therefore, this study was undertaken to optimize and validate the HPLC-DAD-Q-TOF method for the assessment of Nec-1s levels in the plasma what is the necessity for designing its proper dosing regimen for in vivo studies. Benefits of the proposed analytical protocol include: (i) simple sample preparation (precipitation of plasma proteins, evaporation of acetonitrile, reconstitution in mobile phase), (ii) fast, selective and sensitive analysis due to a highly orthogonal LC-MS system providing less than 8 min analysis time, (iii) detection of Nec-1s without any matrix interferences, and quantitation of very low concentration levels of Nec-1s (LLOQ ~ 20 ng/mL), (iv) high reliability of Nec-1s determination with precision and accuracy values meeting the FDA criteria for biomedical analysis. The proposed analytical protocol is suitable for routine use in relevant biological studies, and, in this work, it was successfully applied for monitoring of Nec-1s plasma levels in rats providing reproducible and consistent results. Based on pharmacokinetic features, which can also be assessed due to the results of this study, there will be efforts to perform both acute and chronic in vivo studies and potential clinical safety studies first

Programmed Cell Death in the Left and Right Ventricle of the Late Phase of Post-Infarction Heart Failure

While necroptosis has been shown to contribute to the pathogenesis of post-infarction heart failure (HF), the role of autophagy remains unclear. Likewise, linkage between these two cell death modalities has not been sufficiently investigated. HF was induced by 60-min left coronary occlusion in adult Wistar rats and heart function was assessed 6 weeks later followed by immunoblotting analysis of necroptotic and autophagic proteins in both the left (LV) and right ventricle (RV). HF had no effect on RIP1 and RIP3 expression. PhosphoSer229-RIP3, acting as a pro-necroptotic signal, was increased in LV while deceased in RV of failing hearts. Total MLKL was elevated in RV only. Decrease in pSer555-ULK1, increase in pSer473-Akt and no significant elevation in beclin-1 and LC3-II/I ratio indicated rather a lowered rate of autophagy in LV. No beclin-1 upregulation and decreased LC3 processing also suggested the inhibition of both autophagosome formation and maturation in RV of failing hearts. In contrast, p89 PARP1 fragment, a marker of executed apoptosis, was increased in RV only. This is the first study showing a different signaling in ventricles of the late phase of post-infarction HF, highlighting necroptosis itself rather than its linkage with autophagy in LV, and apoptosis in RV

[研究活動]研究トピックス : 太陽フレアにおける白色光フレアカーネルの時間発展および空間分布

Additional file 1. Additional data on patient characteristics and Triton X-114 fractionation