California Pacific Medical Center Research Institute, San Francisco, CA 94107, USA.
Mol Cell Biochem. 2012 Feb;361(1-2):85-96. doi: 10.1007/s11010-011-1092-y. Epub 2011 Oct 1.
Involvement of the calcineurin/NFAT pathway in transcription of cardiac sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) was demonstrated (Prasad and Inesi, Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011) by upregulation of SERCA2 following calcineurin (CN) activation by cytosolic Ca(2+), and downregulation of SERCA2 following CN inhibition with cyclosporine (CsA) or CN subunits gene silencing. We show here that in cultured cardiac myocytes, competitive engagement of the CN/NFAT pathway is accompanied by downregulation of SERCA2 and Ca(2+) signaling alterations. In fact, SERCA2 downregulation occurs following infection of myocytes with adenovirus vectors carrying luciferase or SERCA1 cDNA under control of NFAT-dependent promoters, but not under control of CMV promoters that do not depend on NFAT. SERCA2 downregulation is demonstrated by comparison with endogenous transcription and protein expression standards such as GAPDH and actin, indicating prominent SERCA2 involvement by the CN/NFAT pathway. Transcription of genes involved in hypertrophy, triggered by adrenergic agonist or by direct protein kinase C (PKC) activation with phorbol 12-myristate 13-acetate (PMA), is also prominently dependent on CN/NFAT. This is demonstrated by CN inhibition with CsA, CN subunits gene silencing with siRNA, displacement of NFAT from CN with 9,10-Dihydro-9,10[1',2']-benzenoanthracene-1,4-dione (INCA-6), and myocyte infection with vectors carrying luciferase cDNA under control of NFAT-dependent promoter. We show here that competitive engagement of the CN/NFAT pathway by endogenous genes involved in hypertrophy produces downregulation of SERCA2, reduction of Ca(2+) transport and inadequate Ca(2+) signaling. It is most interesting that, in the presence of adrenergic agonist, specific protein kinase C (PKC) inhibition with 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gö 6983) prevents development of hypertrophy and maintains adequate SERCA2 levels and Ca(2+) signaling.
钙调神经磷酸酶/NFAT 通路参与心脏肌浆网 Ca2+-ATP 酶（SERCA2）的转录，这是通过细胞溶质 Ca2+ 激活钙调神经磷酸酶（CN），随后用环孢素（CsA）或 CN 亚基基因沉默抑制 CN，观察到 SERCA2 的上调（Prasad 和 Inesi，Am J Physiol Heart Circ Physiol 300(1):H173-H180, 2011）。我们在这里表明，在培养的心肌细胞中，CN/NFAT 通路的竞争结合伴随着 SERCA2 的下调和 Ca2+信号转导的改变。事实上，在感染带有荧光素酶或 SERCA1 cDNA 的腺病毒载体的心肌细胞后，会发生 SERCA2 下调，这些载体的启动子受 NFAT 调控，但不受不依赖于 NFAT 的 CMV 启动子调控。通过与内源性转录物和蛋白质表达标准（如 GAPDH 和肌动蛋白）进行比较，证明了 CN/NFAT 通路对 SERCA2 的显著影响，表明 SERCA2 明显参与了 CN/NFAT 通路。由儿茶酚胺激动剂触发的或通过用佛波醇 12-肉豆蔻酸 13-醋酸酯（PMA）直接激活蛋白激酶 C（PKC）引起的肥大相关基因的转录，也明显依赖于 CN/NFAT。这通过 CsA 抑制 CN、siRNA 基因沉默 CN 亚基、用 9,10-二氢-9,10[1',2']-苯并蒽-1,4-二酮（INCA-6）将 NFAT 从 CN 上置换以及用荧光素酶 cDNA 载体感染心肌细胞来证明，这些载体的启动子受 NFAT 调控。我们在这里表明，通过参与肥大的内源性基因竞争结合 CN/NFAT 通路会导致 SERCA2 下调、Ca2+转运减少和 Ca2+信号转导不足。有趣的是，在儿茶酚胺激动剂存在的情况下，用 3-[1-[3-(二甲基氨基)丙基]-5-甲氧基-1H-吲哚-3-基]-4-(1H-吲哚-3-基)-1H-吡咯并[2,5-d] 二酮（Gö 6983）特异性抑制蛋白激酶 C（PKC）可以防止肥大的发展，并维持足够的 SERCA2 水平和 Ca2+信号转导。
