El-Armouche Ali, Singh Jasmin, Naito Hiroshi, Wittköpper Katrin, Didié Michael, Laatsch Alexander, Zimmermann Wolfram-Hubertus, Eschenhagen Thomas
Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, D-20246, Germany.
J Mol Cell Cardiol. 2007 Sep;43(3):371-6. doi: 10.1016/j.yjmcc.2007.05.021. Epub 2007 Jun 7.
PKCalpha has been shown to be a negative regulator of contractility and PKCalpha gene deletion in mice protected against heart failure. Small interfering (si)RNAs mediate gene silencing by RNA interference (RNAi) and may be used to knockdown PKCalpha in cardiomyocytes. However, transfection efficiencies of (si)RNAs by lipofection tend to be low in primary cells. To address this limitation, we developed an adenoviral vector (AV) driving short hairpin (sh)RNAs against PKCalpha (Ad-shPKCalpha) and evaluated its potential to silence PKCalpha in neonatal rat cardiac myocytes and in engineered heart tissues (EHTs), which resemble functional myocardium in vitro. A nonsense encoding AV (Ad-shNS) served as control. Quantitative PCR and Western blotting showed 90% lower PKCalpha-mRNA and 50% lower PKCalpha protein in Ad-shPKCalpha-infected cells. EHTs were infected with Ad-shPKCalpha on day 11 and subjected to isometric force measurements in organ baths 4 days later. Mean twitch tension was >50% higher in Ad-shPKCalpha compared to Ad-shNS-infected EHTs, under basal and Ca(2+)- or isoprenaline-stimulated conditions. Twitch tension negatively correlated with PKCalpha mRNA levels. In summary, AV-delivered shRNA mediated highly efficient PKCalpha knockdown in cardiac myocytes and improved contractility in EHTs. The data support a role of PKCalpha as a negative regulator of myocardial contractility and demonstrate that EHTs in conjunction with AV-delivered shRNA are a useful model for target validation.
蛋白激酶Cα（PKCalpha）已被证明是心肌收缩力的负调节因子，小鼠体内PKCalpha基因缺失可预防心力衰竭。小干扰（si）RNA通过RNA干扰（RNAi）介导基因沉默，可用于敲低心肌细胞中的PKCalpha。然而，脂质体转染（si）RNA在原代细胞中的转染效率往往较低。为了解决这一局限性，我们构建了一种腺病毒载体（AV），其驱动针对PKCalpha的短发夹（sh）RNA（Ad-shPKCalpha），并评估其在新生大鼠心肌细胞和工程心脏组织（EHT）中沉默PKCalpha的潜力，EHT在体外类似于功能性心肌。编码无义序列的AV（Ad-shNS）作为对照。定量PCR和蛋白质印迹分析显示，Ad-shPKCalpha感染的细胞中PKCalpha-mRNA降低90%，PKCalpha蛋白降低50%。在第11天用Ad-shPKCalpha感染EHT，4天后在器官浴中进行等长力测量。在基础、Ca（2+）或异丙肾上腺素刺激条件下，与Ad-shNS感染的EHT相比，Ad-shPKCalpha感染的EHT平均抽搐张力高出>50%。抽搐张力与PKCalpha mRNA水平呈负相关。总之，腺病毒载体递送的shRNA介导心肌细胞中高效的PKCalpha敲低，并改善了EHT的收缩性。这些数据支持PKCalpha作为心肌收缩力负调节因子的作用，并证明EHT与腺病毒载体递送的shRNA相结合是一种用于靶点验证的有用模型。
