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Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor β (TGFβ) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Gαi2. Here in atrial cells 5 days in ovo,TGFβ increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Gαi2. TGFβ also stimulated Gαi2mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFβ receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Gαi2promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Gαi2promoter activity independent of embryonic age. In 5-day atrial cells, TGFβ stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFβ stimulated both pVent and p3TP-lux. Thus TGFβ exerts opposing effects on parasympathetic response and Gαi2expression by activating different type I TGFβ receptors at distinct stages during cardiac development.

The binding of acetylcholine to receptors in the intact heart causes a decrease in the frequency (chronotropic effect) and force (ionotropic effect) of contraction. The studies reported here demonstrate a chronotropic response of cultured embryonic chicken heart cells to the muscarinic agonist carbamoylcholine. This response is markedly decreased after a 3-hr incubation with 0.1 mM carbamoylcholine. In order to determine whether agonist-induced alterations in muscarinic receptors were responsible for this decrease, we studied the effects of incubation with carbamoylcholine on the binding of the 3H-labeled muscarinic antagonist quinuclidinyl benzilate (QNB) to homogenates of heart cell cultures. [3H]QNB binding to homogenates of cultures of embryonic hearts of chicks 9 days in ovowas characterized and shown to have properties similar to those of muscarinic receptors in intact hearts. Binding was both specific and saturable. [3H]QNB was displaced by muscarinic agonists and antagonists in concentrations consistent with their known potency. Binding was poorly inhibited by the nicotinic antagonist D-tubocurarine. Kinetic analysis of the binding of QNB by muscarinic receptors showed that initially the reaction proceeds by formation of a rapidly reversible complex with a Kdof 1.8 nM, which is converted to a slowly reversible form. These properties of muscarinic receptors in heart cell cultures are strikingly similar to those observed in homogenates of intact hearts. Homogenates of heart cell cultures bound 84 ± 6 fmol (mean ± SD) of QNB per mg of protein. The number of receptors remained stable from day 4 to day 8 in culture. Incubation of cultures with 0.1 mM carbamoylcholine for 3 hr decreased QNB binding by 55%, to 38 ± 5 fmol/mg protein. When cell cultures were first homogenized and then incubated with carbamoylcholine, no decrease in QNB binding sites could be detected. Thus, incubation with carbamoylcholine causes loss of muscarinic binding sites as well as decreased physiologic responsiveness to muscarinic agonists.

One of the major side-effects of the use of HMG CoA reductase inhibitors for the treatment of hypercholesterolemia is the development of myositis and, in some patients undergoing concomitant immunosuppressive treatment, the development of rhabdomyolysis. Experiments outlined in these studies demonstrate that inhibitors of HMG-CoA reductase activity which differ primarily in the substitution of a methyl group for a hydroxyl group have differential effects on both cholesterol levels and cell viability in a striated muscle cell model, the mouse C2-C12myoblast. Thus, concentrations as high as 200 μm of pravastatin had little effect on total cholesterol level while 25 μm of lovastatin decreased cellular cholesterol by over 90%. Simvastatin and lovastatin decreased viability of C2-C12myoblasts by nearly 50% at concentrations as low as 1 and 5 μm, respectively, and decreased viability by almost 90% at 10 and 15 μ respectively. However, 300 μm of pravastatin decreased cell viability by less than 50%. The order of potency for the effects on cell viability was simvastatin>lovastatin>>>pravastatin. The possible relationship between effects on cell viability and the development of myositis is discussed.

HeLa cell mitochondrial proteins have been shown to be the products of two separate protein-synthesizing systems; one, the general cellular mechanism, sensitive to inhibition by cycloheximide, the other, a specific mitochondrial system subject to inhibition by low concentrations of chloramphenicol (Galper, J. B., and J. E. Darnell. 1971. J. Mol. Biol 57:363). Preliminary data have suggested that a mitochondrial N-formyl-methionyl-tRNA (f-Met-tRNA) might be the initiator tRNA in the latter (Galper, J. B., and J. E. Darnell. 1969. Biochem. Biophys. Res. Commun. 34:205; 1971. J. Mol. Biol. 57:363). It is demonstrated here that the synthesis of these endogenous mitochondrial proteins is also subject to inhibition by ethidium bromide and decays with a half-life of 1½–2 h in cultures incubated with low concentrations of this dye. The role of formylated f-Met-tRNA as the initiator tRNA in the synthesis of mitochondrial proteins is supported by data from several experiments. The rates of ethidium bromide inhibition of both the charging of f-Met-tRNA and of the synthesis of mitochondrial proteins are strikingly similar. Inhibition by aminopterin of the formylation of f-Met-tRNA greatly depresses the rate of mitochondrial-specific protein synthesis. In the absence of the synthesis of these proteins, respiration, the levels of cytochromes a–a3 and b, and the number of mitochondrial cristae are decreased. The implications of these findings as they relate to mitochondrial biogenesis are discussed.