Seth M, Sumbilla C, Mullen S P, Lewis D, Klein M G, Hussain A, Soboloff J, Gill D L, Inesi G
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16683-8. doi: 10.1073/pnas.0407537101. Epub 2004 Nov 16.
Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.
胞质Ca2+的瞬时升高是细胞信号传导的常见机制。在横纹肌中，肌浆（内质）网Ca2+ATP酶（SERCA）通过将胞质Ca2+返回细胞内储存库，在终止Ca2+瞬变中起重要作用。然后储存的Ca2+可以再次释放用于后续信号传导。我们通过由外源cDNA模板编码的小干扰RNA的内源性转录，下调培养的心肌细胞中SERCA2基因的表达。cDNA模板由腺病毒载体递送。通过免疫化学、实时RT-PCR和ATP依赖性Ca2+转运的测定，记录了培养物中所有心肌细胞中SERCA表达的降低。SERCA2表达的降低与瞬时受体电位（TRP）通道蛋白（TRPC4和TRPC5）和Na+/Ca2+交换体的上调有关，表明细胞内储存不足通过Ca2+通过质膜的通量得到补偿。事实上，SERCA沉默后，通过钙调神经磷酸酶的激活，Na+/Ca2+交换体、TRPC4、TRPC5和相关转录因子（如刺激蛋白1、心肌细胞增强因子2和活化细胞4的核因子）的转录增加。这一发现表明，观察到的补偿是通过转录串扰和Ca2+信号通路的重塑发生的。这种调节机制的广泛意义与其普遍参与Ca2+信号动力学以及心脏发育和肥大有关。
