Relationship between dibutyryl cyclic AMP and microtubule organization in contracting heart muscle cells.

Abstract

Rhythmic and synchronous contractions of interconnecting myocyte cultures prepared from fetal rat hearts are arrested upon the addition of 0.2-2.0 mM N6,02'-dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP). The contractions arrested by Bt2cAMP are restored either by diluting the Bt2cAMP from the media or by adding colchicine. While colchicine restores Bt2cAMP-arrested myocyte contractions at concentrations as low as 1.0 jIM, the inactive isomer lumicolchicine shows no effect. Morphologically, Bt2cAMP treatment of myocyte cultures results in the appearance of numerous elongated cellular processes not present in control cultures. Ultrastructural examination indicates that in Bt2cAMP-treated cells the intracellular distribution of microtubules is altered such that these organelles appear to accumulate in parallel arrays. In cells not treated with Bt2cAMP, the microtubules appear randomly oriented, while in cells treated with only colchicine, intact microtubules are not observed. The relationship between microtubules and heart cell contraction is discussed. When embryonic heart tissue is dissociated with proteolytic enzymes into single cells and then grown in primary culture, a substantial fraction of the cells display spontaneous and rhythmic contractions (1-4). The time required for the onset of autorhythmicity of primary heart cell cultures is related to the age of the embryo used as the source of the dissociated heart cells (1, 5). A second property of the cultured myocyte system is that the cells of auricular origin contract faster than ventricular cells (1, 6). Thus, the specificity of the compartments of whole heart also manifests itself through the individual cells. Other interesting properties of myocyte cultures are the ability of myocytes to grow independently of each other and beat at individual rates, or the formation of a synchronously beating network of interconnected heart cells achieved by cell contact and possibly multiplication (7,8).The primary myocyte culture has been used as a model for the automaticity and coordinated rhythmic contractile activity of the intact heart with respect to the action of various chronotropic agents such as drugs and hormones (4, 9, 10). In this report, we show that the rhythmic myocyte contraction in culture is reversibly inhibited by 0.2-2.0 mM N6,02'-dibutyryl cyclic AMP (Bt2 cAMP). Colchicine, an inhibitor of microtubule formation, restores the synchronous contraction of Bt2-cAMP-treated myocytes, although the inactive isomer lumicolchicine does not. In addition, this report presents ultrastructural observations indicating that in Bt2cAMP-treated myocyte cultures the intracellular distribution of microtubules is altered such that these organelles appear to accumulate in parallel arrays, while in control cultures the microtubules appear randomly oriented. MATERIALS AND METHODSPreparation of Myocyte Cultures. Minced ventricular muscles dissected from 19-day-old embryos of Sprague-Dawley rats were disaggregated by repeated incubation with 0.25% pancreat...

Ultrastructure of terminally differentiated adult rat cardiac muscle cells in culture

“…Although many were oriented parallel to myofilaments, others intersected myofilament bundles at a variety of angles. Similar microtubule arrangements have been observed in the intact ventricle (Goldstein and Entman, 1979) and neonatal myocyte cultures (Nath et al, 1978).…”

Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents.

“…Although we (5) and others (3,4) have reported that certain tubulin-binding agents stimulate the spontaneous frequency of individual cardiac cells, it was not clear whether this was a general property of all tubulin-binding agents. Our studies here demonstrate across a wide range of compounds that those tubulin-binding agents that depolymerize microtubules (i.e., vinca alkaloids, colchicine, nocodazol, and podophylotoxin) have very early or immediate effects on heart cell beating.…”

“…There have been, however, three scattered reports (3)(4)(5), one of which comes from the laboratory of T.J.L. (5), suggesting that tubulin-binding agents can affect the rate of spontaneously beating cardiac cells growing in vitro (3)(4)(5).…”

“…(5), suggesting that tubulin-binding agents can affect the rate of spontaneously beating cardiac cells growing in vitro (3)(4)(5).…”

Properties of a microtubule-associated cofactor-independent protein kinase from pig brain

“…Each enzyme phosphorylates MAP2 in vitro, decreasing the ability of MAP2 to promote microtubule assembly (Yamamoto et al, 1985). Since cAMP and Ca2" plus calmodulin effect the number and organization of microtubules in intact cells (Kram & Tomkins, 1973;Nath et al, 1978;Keith et al, 1983), it is possible that phosphorylation of MAPs may be one mechanism for modulating microtubule function.…”