Liu Weiran, Yasui Kenji, Opthof Tobias, Ishiki Ryoji, Lee Jong-Kook, Kamiya Kaichiro, Yokota Mitsuhiro, Kodama Itsuo
Department of Circulation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan.
Life Sci. 2002 Aug 2;71(11):1279-92. doi: 10.1016/s0024-3205(02)01826-x.
Transplant of immature cardiomyocytes is recently attracting a great deal of interest as a new experimental strategy for the treatment of failing hearts. Full understanding of normal cardiomyogenesis is essential to make this regenerative therapy feasible. We analyzed the molecular and functional changes of Ca(2+) handling proteins during development of the mouse heart from early embryo at 9.5 days postcoitum (dpc) through adulthood. From the early to the late (18 dpc) embryonic stage, mRNAs estimated by the real time PCR for ryanodine receptor (type 2, RyR2), sarcoplasmic reticulum (SR) Ca(2+) pump (type 2, SERCA2) and phospholamban (PLB) increased by 3-15 fold in the values normalized to GAPDH mRNA, although Na(+)/Ca(2+) exchanger (type 1, NCX1) mRNA was unchanged. After birth, there was a further increase in the mRNAs for RyR2, SERCA2 and PLB by 18-33 fold, but a 50% decrease in NCX1 mRNA. The protein levels of RyR2, SERCA2, PLB and NCX1, which were normalized to total protein, showed qualitatively parallel developmental changes. L-type Ca(2+) channel currents (I(Ca-L)) were increased during the development (1.3-fold at 18 dpc, 2.2-fold at adult stage, vs. 9.5 dpc). At 9.5 dpc, the Ca(2+) transient was, unlike adulthood, unaffected by the SR blockers, ryanodine (5 microM) and thapsigargin (2 microM), and also by a blocker of the Ca(2+) entry via Na(+)/Ca(2+) exchanger, KB-R 7943 (1 microM). The Ca(2+) transient was abolished after application of nisoldipine (5 microM). These results indicate that activator Ca(2+) for contraction in the early embryonic stage depends almost entirely on I(Ca-L).
未成熟心肌细胞移植作为治疗衰竭心脏的一种新的实验策略，近来引起了广泛关注。充分了解正常心肌发生对于使这种再生疗法可行至关重要。我们分析了从妊娠9.5天（dpc）的早期胚胎到成年期小鼠心脏发育过程中钙（Ca(2+)）处理蛋白的分子和功能变化。从胚胎早期到晚期（18 dpc），经实时PCR估算，与GAPDH mRNA标准化后，兰尼碱受体（2型，RyR2）、肌浆网（SR）钙（Ca(2+)）泵（2型，SERCA2）和受磷蛋白（PLB）的mRNA增加了3至15倍，而钠（Na(+)/钙（Ca(2+)）交换体（1型，NCX1）mRNA无变化。出生后，RyR2、SERCA2和PLB的mRNA进一步增加了18至33倍，但NCX1 mRNA减少了50%。以总蛋白标准化后的RyR2、SERCA2、PLB和NCX1的蛋白水平显示出定性的平行发育变化。L型钙（Ca(2+)）通道电流（I(Ca-L)）在发育过程中增加（18 dpc时为1.3倍，成年期为2.2倍，相对于9.5 dpc）。在9.5 dpc时，与成年期不同，钙（Ca(2+)）瞬变不受SR阻滞剂兰尼碱（5 microM）和毒胡萝卜素（2 microM）以及通过钠（Na(+)/钙（Ca(2+)）交换体的钙（Ca(2+)）内流阻滞剂KB-R 7943（1 microM）的影响。应用尼索地平（5 microM）后钙（Ca(2+)）瞬变消失。这些结果表明，胚胎早期收缩的激活钙（Ca(2+)）几乎完全依赖于I(Ca-L)。
