Cardionome Laboratory, Department of Biomedical Sciences, Texas A&M Health Science Center Baylor College of Dentistry, Dallas, TX, USA.
Br J Pharmacol. 2010 May;160(1):93-100. doi: 10.1111/j.1476-5381.2010.00686.x. Epub 2010 Mar 19.
The present study tested the hypothesis that selective caspase-3 (C-3) knock-out would regulate the contractile actions of noradrenaline (NA) in the dysfunction of adult rat ventricular myocytes (ARVMs) induced by sepsis. Here, we have studied the contractile response of ARVMs, transfected with C-3 small interfering RNA (C-3 siRNA), to NA.
Single ARVMs were isolated from the hearts of male Sprague-Dawley rats 3 days after induction of sepsis, and from sham-treated rats. The sham and septic ARVMs were treated with NA (10 microM) alone or after transfection with C-3 siRNA or non-silencing RNA (2 microM). Mechanical properties were measured digitally, and immunoblotting and immunocytochemical analyses were carried out.
The NA-induced increase in peak shortening (PS) was less in septic ARVMs and transfection with C-3 siRNA produced a significant increase in this PS. Immunocytochemical and immunoblot analyses revealed that NA exacerbated sepsis-induced up-regulation of C-3. Transfection of septic ARVMs with C-3 siRNA exhibited a decreased expression of C-3 fluorescence after NA. In septic ARVMs, we also observed a down-regulation of contractile proteins (alpha-actin, myosin light chain-1 and tropomyosin) along with DNA damage. Transfection of septic ARVMs with C-3 siRNA produced an increase in the expression of contractile proteins, and a decrease in DNA damage.
These data suggest that C-3 knock-down improved the loss of contractile response to NA in septic ARVMs, suggesting that C-3 regulated contractile dysfunction induced by sepsis in ARVMs.
本研究旨在验证选择性半胱氨酸天冬氨酸蛋白酶-3（C-3）敲除是否会调节脓毒症诱导的成年大鼠心室肌细胞（ARVM）功能障碍中去甲肾上腺素（NA）的收缩作用。在这里，我们研究了转染 C-3 小干扰 RNA（C-3 siRNA）的 ARVM 对 NA 的收缩反应。
在脓毒症诱导后 3 天，从雄性 Sprague-Dawley 大鼠心脏中分离出单 ARVM，并从假手术处理的大鼠心脏中分离出单 ARVM。用 NA（10 μM）单独处理或用 C-3 siRNA 或非沉默 RNA（2 μM）转染后处理 sham 和脓毒症 ARVM。数字测量机械性能，并进行免疫印迹和免疫细胞化学分析。
NA 诱导的峰值缩短（PS）增加在脓毒症 ARVM 中较少，而转染 C-3 siRNA 可显著增加 PS。免疫细胞化学和免疫印迹分析显示，NA 加剧了脓毒症诱导的 C-3 上调。转染 C-3 siRNA 的脓毒症 ARVM 在 NA 后表现出 C-3 荧光表达降低。在脓毒症 ARVM 中，我们还观察到收缩蛋白（α-肌动蛋白、肌球蛋白轻链-1 和原肌球蛋白）的表达下调以及 DNA 损伤。转染脓毒症 ARVM 的 C-3 siRNA 可增加收缩蛋白的表达，减少 DNA 损伤。
这些数据表明，C-3 敲低改善了脓毒症 ARVM 对 NA 收缩反应的丧失，表明 C-3 调节了 ARVM 中脓毒症引起的收缩功能障碍。
