Andrews Catherine, Ho Peter D, Dillmann Wolfgang H, Glembotski Christopher C, McDonough Patrick M
Genetics Cell Biology/Development, University of Minnesota, 1445 Gortner Ave, St. Paul, MN 55108, USA.
Cardiovasc Res. 2003 Jul 1;59(1):46-56. doi: 10.1016/s0008-6363(03)00329-8.
Our goal was to determine if the MKK6-p38 MAPK pathway regulates cardiac intracellular calcium (Ca(2+)). We also tested if MKK6 might influence expression of SERCA2, a calcium regulatory molecule involved in relaxation, and the activity of nuclear factor of activated T-cells (NF-AT), a calcium-regulated transcription factor that participates in pathological responses to pressure-overload.
Neonatal rat ventricular myocytes were transfected with MKK6(Glu), an activator of p38 MAPK. Green fluorescent protein (GFP) was used as transfection marker and Ca(2+) was evaluated via indo-1. SERCA2 expression was assayed via Northern and Western techniques. The activity of the rat SERCA2 gene promoter and NF-AT-dependent gene expression were monitored with reporter genes. Myocyte contractility was regulated by electrical pacing.
MKK6(Glu) prolonged decay of the contractile calcium transients, downregulated SERCA2 expression, and reduced the activity of the rat SERCA2 gene promoter. Diastolic Ca(2+) in myocytes pacing at 1-2 Hz was dramatically increased by MKK6(Glu). NF-AT-dependent gene expression was activated by MKK6(Glu) and by pacing of contractions in a synergistic manner. Overexpression of SERCA2 mitigated the effects of MKK6(Glu) on Ca(2+) and NF-AT.
The MKK6(Glu)-p38 MAPK pathway prolongs the decay phase of the cardiac contractile calcium by downregulating SERCA2, increasing diastolic Ca(2+) which activates NF-AT. The ability of SERCA2 over-expression to reduce NF-AT activity represents a potential novel therapeutic effect of SERCA2 that should be further considered in the development of cardiac gene therapy strategies.
我们的目标是确定MKK6-p38丝裂原活化蛋白激酶（MAPK）信号通路是否调节心脏细胞内钙浓度（Ca(2+)）。我们还测试了MKK6是否可能影响肌浆网钙ATP酶2（SERCA2）的表达，SERCA2是一种参与舒张的钙调节分子，以及活化T细胞核因子（NF-AT）的活性，NF-AT是一种参与压力超负荷病理反应的钙调节转录因子。
用p38 MAPK的激活剂MKK6(Glu)转染新生大鼠心室肌细胞。绿色荧光蛋白（GFP）用作转染标记物，通过indo-1评估Ca(2+)。通过Northern和Western技术检测SERCA2的表达。用报告基因监测大鼠SERCA2基因启动子的活性和NF-AT依赖性基因表达。通过电起搏调节心肌细胞收缩力。
MKK6(Glu)延长了收缩期钙瞬变的衰减，下调了SERCA2的表达，并降低了大鼠SERCA2基因启动子的活性。在1-2 Hz起搏的心肌细胞中，MKK6(Glu)使舒张期Ca(2+)显著增加。MKK6(Glu)和收缩期起搏以协同方式激活NF-AT依赖性基因表达。SERCA2的过表达减轻了MKK6(Glu)对Ca(2+)和NF-AT的影响。
MKK6(Glu)-p38 MAPK信号通路通过下调SERCA2延长心脏收缩期钙的衰减期，增加舒张期Ca(2+)，进而激活NF-AT。SERCA2过表达降低NF-AT活性的能力代表了SERCA2潜在的新治疗作用，在心脏基因治疗策略的开发中应进一步考虑。
