Conway S J, Godt R E, Hatcher C J, Leatherbury L, Zolotouchnikov V V, Brotto M A, Copp A J, Kirby M L, Creazzo T L
Developmental Biology Program, Institute of Molecular Medicine and Genetics, Augusta, GA 30912-2640, USA.
J Mol Cell Cardiol. 1997 Oct;29(10):2675-85. doi: 10.1006/jmcc.1997.0499.
Around 85% of embryos homozygous for the splotch (Sp2H) allele (Sp2H/Sp2H), a Pax3 mutation, develop persistent truncus arteriosus (PTA), a defect related to the cardiac neural crest. These embryos die by 14.5 days post coitum. In an investigation of the cause of lethality in these embryos, we used digital video imaging microscopy to examine beating embryonic hearts in situ at 13.5 dpc. The hearts of Sp2H/Sp2H embryos with PTA clearly showed poor function when compared with normal litter mates. Contractile force was examined in detergent-skinned ventricular muscle strips from Sp2H/Sp2H embryos at ages 12.5 and 13.5 dpc. There was no significant difference in the maximum force or in myosin content between Sp2H/Sp2H and control groups, indicating no significant dysfunction of the contractile apparatus in hearts from Sp2H/Sp2H embryos. Ca2+ transients were examined in enzymatically-dissociated ventricular myocytes and were significantly reduced in defective hearts, indicating that reduced cardiac function in Sp2H/Sp2H embryos with PTA was due to impaired excitation-contraction (EC) coupling. Ca2+ currents were examined using the perforated patch clamp technique. The magnitude of the Ca2+ current was found to be reduced by approximately 3.2-fold in Sp2H/Sp2H hearts with PTA compared to normal. Since the sarcoplasmic reticulum is sparse or absent in the embryonic heart, the impaired EC coupling was due to the reduction in Ca2+ current. These observations suggest that neural crest abnormalities result in a defect in EC coupling, causing depressed myocardial function and death in utero from cardiac failure. Interestingly, Sp2H/Sp2H hearts without PTA had normal EC coupling. These results indicated that impaired EC coupling was secondary to the Pax3 mutation. The findings in this report indicate an important role for the neural crest in the development of normal myocardial function, and represent the first demonstration of impaired excitation-contraction coupling in a genetically-defined embryonic mammalian model of a cardiac structural defect.
大约85% 携带斑点（Sp2H）等位基因（Sp2H/Sp2H）纯合子的胚胎（一种Pax3突变）会发育出永存动脉干（PTA），这是一种与心脏神经嵴相关的缺陷。这些胚胎在交配后14.5天死亡。在对这些胚胎致死原因的调查中，我们使用数字视频成像显微镜在胚胎发育13.5天时原位检查跳动的胚胎心脏。与正常同窝仔相比，患有PTA的Sp2H/Sp2H胚胎的心脏功能明显较差。在胚胎发育12.5天和13.5天时，对来自Sp2H/Sp2H胚胎的经去污剂处理的心室肌条的收缩力进行了检测。Sp2H/Sp2H组和对照组之间在最大力或肌球蛋白含量上没有显著差异，表明Sp2H/Sp2H胚胎心脏的收缩装置没有明显功能障碍。在酶解离的心室肌细胞中检测了Ca2+瞬变，在有缺陷的心脏中Ca2+瞬变显著降低，表明患有PTA的Sp2H/Sp2H胚胎心脏功能降低是由于兴奋 - 收缩（EC）偶联受损。使用穿孔膜片钳技术检测了Ca2+电流。发现与正常心脏相比，患有PTA的Sp2H/Sp2H心脏中Ca2+电流的幅度降低了约3.2倍。由于胚胎心脏中的肌浆网稀疏或不存在，EC偶联受损是由于Ca2+电流减少所致。这些观察结果表明，神经嵴异常导致EC偶联缺陷，引起心肌功能抑制和子宫内因心力衰竭死亡。有趣的是，没有PTA的Sp2H/Sp2H心脏具有正常的EC偶联。这些结果表明，EC偶联受损是Pax3突变的继发结果。本报告中的发现表明神经嵴在正常心肌功能发育中起重要作用，并且代表了在基因定义的心脏结构缺陷胚胎哺乳动物模型中首次证明兴奋 - 收缩偶联受损。
