Alyonycheva T, Cohen-Gould L, Siewert C, Fischman D A, Mikawa T
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021, USA.
Circ Res. 1997 May;80(5):665-72. doi: 10.1161/01.res.80.5.665.
Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.
心脏收缩是由电兴奋通过心脏传导系统的特殊组织进行传导来协调的。通过对胚胎鸡心脏进行逆转录病毒单细胞标记和谱系分析，我们最近证明，一部分心肌细胞最终分化为外周传导系统（浦肯野纤维）的细胞，而且这种情况总是发生在发育中的冠状动脉的血管周围区域。在传导系统中发挥转录调控作用的顺式调控元件已与收缩性心肌细胞中的元件区分开来；例如，结蛋白基因的5'调控序列在骨骼肌和传导系统中作为增强子元件起作用，但在心肌中不起作用。我们假设浦肯野纤维的分化涉及基因表达程序从心肌特征性的程序转变为骨骼肌典型的程序。为了验证这一假设，我们检测了鸡心脏浦肯野纤维中肌球蛋白结合蛋白-H（MyBP-H）的表达。这种独特的肌球蛋白结合蛋白存在于骨骼肌细胞而非心肌细胞中。我们制备了针对MyBP-H的位点定向多克隆抗体（AB105）。对心肌的免疫组织学分析将AB105抗原主要定位到浦肯野纤维内肌原纤维的A带。对成年鸡心脏心室壁全提取物的蛋白质印迹分析表明，AB105表位仅限于一种大小约为86 kD的单一蛋白质，与骨骼肌中的MyBP-H大小相同。浦肯野纤维86-kD蛋白质的生化特性及其mRNA的核糖核酸酶保护分析表明，浦肯野纤维86-kD蛋白质与骨骼肌MyBP-H无法区分。这些结果提供了证据，表明骨骼肌MyBP-H在分化为心脏浦肯野纤维的一部分心肌细胞中表达。
