Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.
Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.
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COS-1细胞和心肌细胞中SERCA1和SERCA2a表达的比较。
DOI：
10.1152/ajpheart.1999.277.6.h2381
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Inesi,G
中科院分区：
文献类型：
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作者：
Sumbilla,C;Cavagna,M;Zhong,L;Ma,H;Lewis,D;Farrance,I;Inesi,G
培养的COS-1细胞，以及鸡胚和新生大鼠心肌细胞，感染重组腺病毒载体，以确定限制因素的表达和重组肌质内质网钙（SERCA）异构体的钙转运功能。滴度实验表明，所有COS-1细胞和培养的心肌细胞可以感染的腺病毒滴度为10空斑形成单位（pfu）每个接种细胞。提高腺病毒滴度进一步产生更高的蛋白质表达，达到功能性膜结合SERCA蛋白的渐近极限。SERCA表达的渐近行为与转录无关，而是由于转录后事件。最小（−268）心肌肌钙蛋白T（cTnT）启动子是腺病毒载体构建的方便尺寸，并表现出紧密肌肉特异性。然而，其效率低于非特异性巨细胞病毒（CMV）启动子。无论如何，只要调整病毒滴度以补偿转录效率，使用CMV和cTnT启动子就可以获得相同的SERCA表达的最大水平。达到了总SERCA蛋白表达超过对照肌细胞的内源性SERCA水平的最大三倍增加（由于感染后内源性SERCA的减少，与相同感染的肌细胞的内源性SERCA相比增加了七倍）。与先前的报道[Ji等人Am. J. Physiol.276（Heart Circ.生理学45）：H89-H97，1999]，与SERCA 2a同种型相比，证明了SERCA 1具有更高的动力学转换，如分别伴随重组SERCA 1或SERCA 2a的最大表达的钙摄取速率增加5.0倍对2.6倍所示。这一信息被认为是必要的研究试图修改心肌细胞功能的操纵SERCA的表达。
Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca2+(SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (−268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al.Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89–H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.