Alam Hasan B, Shults Christian, Ahuja Naresh, Ayuste Eduardo C, Chen Huazhen, Koustova Elena, Sailhamer Elizabeth A, Li Yongqing, Liu Baoling, de Moya Marc, Velmahos George C
Trauma Research and Readiness Institute for Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, United States.
Resuscitation. 2008 Feb;76(2):299-310. doi: 10.1016/j.resuscitation.2007.07.030. Epub 2007 Sep 5.
Chromatin remodeling through histone acetylation is a key control mechanism in gene transcription. We have shown previously that fluid resuscitation in rodents is coupled with highly structured post-translational modifications of cardiac histones. The current experiment was performed to validate this concept in a clinically relevant large animal model of hemorrhage and resuscitation, and to correlate the changes in histone acetylation with altered expression of immediate-early response genes.
Yorkshire swine (n=49, 7/group, weight=40-58kg) were subjected to combined uncontrolled and controlled hemorrhage (40% of estimated blood volume) and randomly assigned to the following resuscitation groups: (1) 0.9% saline (NS), (2) racemic lactated Ringer's (dl-LR), (3) l-isomer lactated Ringer's (l-LR), (4) Ketone Ringer's (KR), (5) 6% hetastarch in saline (Hespan). KR contained an equimolar substitution of lactate with beta-hydroxybutyrate. No hemorrhage (NH) and no resuscitation (NR) groups were included as controls. Cardiac protein was used in Western blotting to analyze total protein acetylation and histone acetylation specifically. Lysine residue-specific acetylation of histone subunits H3 and H4 was further evaluated. In addition, Chromatin Immunoprecipitation (ChIP) technique was used to separate the DNA bound to acetylated histones (H3 and H4 subunits), followed by measurement of genes that are altered by hemorrhage/resuscitation, including immediate-early response genes (c-fos and c-myc), and heat shock protein (HSP) 70.
The type of fluid used for resuscitation influenced the patterns of cardiac histone acetylation. Resuscitation with dl-LR and KR induced hyperacetylation on H3K9. KR resuscitation was also associated with increased acetylation on H3K14 and H4K5, and hypoacetylation on H3K18. The expression of genes was also fluid specific, with the largest number of changes following KR resuscitation (increased c-fos and c-myc, HSP 70 linked with H3; and increased c-myc linked with H4). Among the histone subunits studied, altered H3 acetylations were associated with the majority of changes in immediate-early gene expression.
Acetylation status of cardiac histones, affected by hemorrhage, is further modulated by resuscitation producing a fluid-specific code that is preserved in different species. Resuscitation with KR causes histone acetylation at the largest number of lysine sites (predominately H3 subunit), and has the most pronounced impact on the transcriptional regulation of selected (immediate-early response) genes.
通过组蛋白乙酰化进行的染色质重塑是基因转录中的关键调控机制。我们之前已经表明，啮齿动物的液体复苏与心脏组蛋白高度结构化的翻译后修饰相关。当前实验旨在在临床相关的大出血和复苏大型动物模型中验证这一概念，并将组蛋白乙酰化的变化与即刻早期反应基因表达的改变相关联。
对约克夏猪（n = 49，每组7只，体重 = 40 - 58kg）进行联合非控制性和控制性出血（估计血容量的40%），并随机分配到以下复苏组：（1）0.9%生理盐水（NS），（2）消旋乳酸林格氏液（dl-LR），（3）左旋乳酸林格氏液（l-LR），（4）酮林格氏液（KR），（5）6%羟乙基淀粉生理盐水（贺斯）。KR含有等量的β-羟基丁酸盐替代乳酸盐。未出血（NH）和未复苏（NR）组作为对照。使用心脏蛋白进行蛋白质免疫印迹分析，以特异性分析总蛋白乙酰化和组蛋白乙酰化。进一步评估组蛋白亚基H3和H4的赖氨酸残基特异性乙酰化。此外，使用染色质免疫沉淀（ChIP）技术分离与乙酰化组蛋白（H3和H4亚基）结合的DNA，随后测量因出血/复苏而改变的基因，包括即刻早期反应基因（c-fos和c-myc）以及热休克蛋白（HSP）70。
用于复苏的液体类型影响心脏组蛋白乙酰化模式。用dl-LR和KR复苏诱导H3K9高乙酰化。KR复苏还与H3K14和H4K5乙酰化增加以及H3K18低乙酰化相关。基因表达也是液体特异性的，KR复苏后变化数量最多（c-fos和c-myc增加，HSP 70与H3相关；c-myc增加与H4相关）。在所研究的组蛋白亚基中，H3乙酰化改变与即刻早期基因表达的大多数变化相关。
受出血影响的心脏组蛋白乙酰化状态通过复苏进一步调节，产生一种在不同物种中都存在的液体特异性编码。用KR复苏导致在最多的赖氨酸位点（主要是H3亚基）发生组蛋白乙酰化，并且对选定的（即刻早期反应）基因的转录调控影响最为显著。
