Prasad A M, Ma H, Sumbilla C, Lee D I, Klein M G, Inesi G
California Pacific Medical Center Research Institute, 475 Brannan St., San Francisco, CA 94107, USA.
Am J Physiol Cell Physiol. 2007 Jun;292(6):C2269-75. doi: 10.1152/ajpcell.00441.2006. Epub 2007 Feb 7.
We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.
我们致力于使用一个基础且可控的实验系统，来描述肌浆（内质）网Ca(2+) -ATP酶（SERCA）在心肌肥大发展过程中的参与程度和时间顺序，包括转录、蛋白质表达、Ca(2+)转运以及细胞质Ca(2+)信号传导。为此，在用去氧肾上腺素（PE）进行肾上腺素能激活后，获得了培养的新生大鼠心肌细胞肥大模型。通过显微图像评估心肌细胞大小、[(14)C]苯丙氨酸掺入量和总蛋白表达的增加，以及心房利钠肽转录的增加，明确证实了肥大的发生。肥大的一个早期且显著的特征是SERCA2转录本减少，这是通过参照甘油醛-3-磷酸脱氢酶等稳定标记物进行RT-PCR测定得出的。Ca(2+)-ATP酶蛋白水平和Ca(2+)转运活性降低至对照值的约50%，这一过程有一定延迟，显然是对转录产生初级效应的结果。在电刺激后，用Ca(2+)敏感染料测量的细胞质Ca(2+)信号动力学在肥大心肌细胞中发生了显著改变。然而，PE诱导的肥大对细胞质Ca(2+)信号动力学的影响不如用小干扰RNA使SERCA2大幅下调或用毒胡萝卜素（10 nM）抑制时在心肌细胞中观察到的那样显著。我们得出结论，肥大刺激后SERCA2会发生显著下调，这可能是由于肥大转录程序未涉及SERCA基因所致。SERCA2下调对Ca(2+)信号传导的影响部分被其他Ca(2+)转运机制所补偿。这些改变可能导致长期肥大中功能衰竭的逐渐发生。
