Anat Embryo1 (1989) 179:387 393 Anatomy and Embryology 9 Springer-Verlag 1989 Creatine kinase isozyme expression in embryonic chicken heart Wouter H. Lamers 1, Willie J.C. Geerts I, Antoon F.M. Moorman 1, and Robert P. Dottin 2 1 Department of Anatomy and Embryology, University of Amsterdam, Meibergdreef t 5, 1105 AZ Amsterdam, The Netherlands 2 Department of Biological Sciences, Hunter College, CUNY, 695 Park Avenue, New York, NY 10211, USA Summary. The distribution pattern of creatine kinase (EC 2.7.3.2) isozymes in developing chicken heart was stud- ied by immunohistochemistry. Creatine kinase M, which is absent from adult heart, is transiently expressed between 4 and 11 days of incuba- tion. During that period, numerous muscular cells in the roof and septum of the atrium, in the interventricular sep- turn and on top of the trabeculae cordis and at the rim of the outflow tract stain strongly with a polyclonal anti- body that is specific for the M subunit. In the ventricle and outflow tract, the M-positive cells are found mainly subendocardially and in the right half, at the transition of conducting and working myocytes. Creatine kinase B, which is the predominant adult isozyme, is initially expressed to a high concentration in a small group of disperse myocardial cells in the upstream part of the inflow tract. When compared to the expression pattern of cardiac myosin heavy chains, the observed creatine kinase expres- sion pattern suggests that M-positive cells are mainly found in areas that participate in the formation of cardiac conduc- tive tissue, whereas B-positive cells are first found in areas that are involved in the generation of cardiac rhythm. Key words: Distribution pattern - Creatine kinase isozymes - Embryonic chicken heart - Immunohistochemistry Indroduetion A very economical mechanism for energy transfer may be required for several energy consuming processes in excitable tissues, such as muscle and neural tissue (for a discussion: see Kammermeier 1987b). Recent experiments and calcula- tions show that the phosphocreatine shuttle between mito- chondria and cytosolic microcompartments represents an intracellular mechanism to assure a high efficiency of energy transduction via ATP. The shuttle mechanism requires the presence of creatine kinases (CK, E.C. 2.7.3.2) at the site of ATP production (" mitochondrial" CK) and at the site of consumption ("cytosolic" CK) (for a review, see Bess- man 1985, 1987). In all species two different but related cytosolic gene products, designated '~ (for muscle) and "B" (for brain) and one unrelated, mitochondrial gene product (designated "Mi") are found (for a review, see Babbitt et al. 1986). The enzyme exists as a dimer. In the cytosol 3 different isozyme are found, MM, BB and the Offprint requests to: W.H. Lamers hybrid MB. The MM isozyme is prevalent in mature skele- tal muscle and mammalian myocardium, whereas the BB isozyme is prevalent in neural tissue and avian myocardium (Eppenberger et al. 1967). Only adult mammalian myocar- dium contains appreciable amounts of the MB isozyme (Roberts et al. 1975). Creatine kinase enzyme levels increase relatively late in organ development (Ziter 1974; Hall and De Luca 1973; Ingwall etal. 1981; Eppenberger etal. 1964, 1983; Robin- son 1987; Perriard et al. 1987). Initially all organs that con- tain CK, express low levels of the BB isozyme. In avian and mammalian skeletal muscle and in mammalian heart, CK BB is subsequently replaced by the CK MM isozyme. The hybrid enzyme CK MB is formed during the transition- al period (Ziter 1974; Hall and DeLuca 1973; Perriard et al. 1978). Despite pronounced changes in isozyme expression with development, very few studies have focussed on the spatial distribution of the expression patterns of the respective isozymes during the transitional period. We have recently studied the distribution patterns of atrial and ventricular myosin heavy chains in developing chicken heart (Sanders et al. 1986; de Groot et al./987; de Jong et al. 1987). These studies showed that prior to its morphological differentia- tion, the developing conductive tissue is characterized by a coexpression of atrial and ventricular myosin heavy chains. In view of our interest in the origin of the cardiac conductive tissue, it appeared of interest to look at the development of the distribution pattern of CK isozymes in embryonic chicken hearts. Materials and methods Animals White Leghorn chicken embryos of 3-13 days of incubation were staged according to Hamburger and Hamilton (1951) and fixed at room temperature for 2 h in a mixture of meth- anol, acetone, acetic acid and water (9:9:2:4 by vol.). After dehydration in dimethoxypropane and embedding in parap- last (Paraplast Plus, Lancer, Oxford), serial sections of the embryos were prepared for immunohistochemistry. Antibodies The isolation of the creatine kinase BB and MM isozymes from chicken heart (BB) and pectoralis muscle (MM), re- spectively, and the preparation and characterization of the polyclonal antibodies have been described (Schweinfest