Background

The severity and duration of hypoxia is known to determine apoptotic fate in heart, however, its implication during myocardial infarction (MI) remains unaddressed. Therefore the aim of the study was to determine apoptotic regulation in cardiomyocytes under varied hypoxic intensity and duration and to unravel the role of HIF-1α in such modulation.

Methods

Treatment of cardiomyocytes to varied hypoxic intensity and duration was carried out in vitro, which was mimicked in vivo by dose-dependent Isoproterenol hydrochloride treatment for varied time-points. Myocardium-targeted HIF-1α knockdown in vivo was performed to decipher its role in cardiomyocyte apoptosis under varied stress. Signaling intermediates were analyzed by RT-PCR, immunoblotting and co-immunoprecipitation. DCFDA-based ROS assay, Griess assay for NO release and biochemical assays for estimating caspase activity were performed.

Results

Severe stress resulted in cardiomyocyte apoptosis in both shorter and longer time-points. Moderate stress, on the other hand, induced apoptosis only in the shorter time-point which was downregulated in the longer time-point. ROS activity was upregulated under severe hypoxic stress for both time-points and only in the early time-point under moderate hypoxia. Increased ROS accumulation activated ERK-1/2 which stabilized nuclear HIF-1α, promoting bnip3-mediated apoptosis. Stable HSP90-IRE-1 association in such cells caused elevated endoplasmic reticulum-stress-related caspase-12 activity. Sustained moderate hypoxia caused decline in ROS activity, but upregulated NFκB-dependent NO generation. NO-stabilized HIF-1α was predominantly cytosolic, since low ROS levels downregulated ERK-1/2 activity, thereby suppressing bnip3 expression. Cytosolic HIF-1α in such cells sequestered HSP90 from IRE-1, downregulating caspase-12 activity due to proteasomal degradation of IRE-1. Accordingly, myocardium-specific in vivo silencing of HIF-1α was beneficial at both time-points under severe stress as also for lesser duration of moderate stress. However, silencing of HIF-1α aggravated apoptotic injury during sustained moderate stress.

Conclusion

ROS-mediated HIF-1α stabilization promotes cardiomyocyte apoptosis on one hand while NO-mediated stabilization of HIF-1α disrupts apoptosis depending upon the severity and duration of hypoxia. Therefore the outcome of modulation of cardiac HIF-1α activity is regulated by both the severity and duration of ischemic stress.

中文翻译：

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低氧应激的程度和持续时间差异性调节心肌梗死期间HIF-1α介导的心肌细胞凋亡信号环境。

背景

已知缺氧的严重程度和持续时间决定着心脏的凋亡命运，但是，其在心肌梗塞（MI）期间的影响仍未得到解决。因此，该研究的目的是确定在不同的低氧强度和持续时间下心肌细胞的凋亡调节，并阐明HIF-1α在这种调节中的作用。

方法

在体外对心肌细胞进行不同程度的低氧强度和持续时间的治疗，在体内通过剂量依赖性盐酸异丙肾上腺素在不同的时间点进行模拟。进行了体内靶向心肌的HIF-1α敲低，以研究其在各种压力下在心肌细胞凋亡中的作用。通过RT-PCR，免疫印迹和免疫共沉淀法分析信号中间体。进行了基于DCFDA的ROS测定，用于NO释放的Griess测定和用于评估caspase活性的生化测定。

结果

严重的压力导致在较短和较长时间点的心肌细胞凋亡。另一方面，适度的压力仅在较短的时间点诱导凋亡，而在较长的时间点被下调。在严重低氧应激的两个时间点和仅在中度低氧下的早期时间点，ROS活性上调。ROS积累的增加激活了ERK-1 / 2，从而稳定了核HIF-1α，促进了bnip3介导的细胞凋亡。此类细胞中稳定的HSP90-IRE-1缔合导致内质网应激相关的caspase-12活性升高。持续的中度缺氧会引起ROS活性下降，但会上调NFκB依赖性NO的产生。NO稳定的HIF-1α主要是胞质的，因为低ROS水平会下调ERK-1 / 2活性，从而抑制bnip3表达式。此类细胞中的胞质HIF-1α从IRE-1中分离出HSP90，由于IRE-1的蛋白酶体降解而下调caspase-12活性。因此，HIF-1α的心肌特异性体内沉默在重度压力下的两个时间点都是有益的，对中度压力的持续时间也较短。但是，HIF-1α沉默会在持续的中等压力下加剧细胞凋亡。

结论

ROS介导的HIF-1α稳定一方面促进了心肌细胞的凋亡，而NO介导的HIF-1α稳定则根据缺氧的严重程度和持续时间破坏了细胞凋亡。因此，心脏HIF-1α活性的调节结果受缺血性应激的严重程度和持续时间的调节。