Akiko Arai

Murine cardiac progenitor cells require visceral embryonic endoderm and primitive streak for terminal differentiation

Cardiac progenitor cells in avian and amphibian embryos are known to commit to cardiac lineage during gastrulation or early neurulation. These cells require cell interaction with anterior endoderm for their differentiation into cardiomyocytes. However, little is known about cell interaction in mammalian cardiogenesis. We investigated the staging of murine cardiomyocyte commitment and the role of cell interaction in differentiation of cardiac progenitor cells into cardiomyocytes, using cultures of various embryonic regions at 7.25 and 7.5 days post coitum (p.c.), respectively. To evaluate the terminal differentiation of cardiac progenitor cells, we employed three parameters; expression of spontaneous beating, myosin heavy chain (MHC) protein, and cardiac‐specific genes (α myosin heavy chain, Csx/Nkx2.5 and myosin light chain 2V genes). mRNAs of cardiac‐specific genes were detected in 7.25‐day p.c. mesoderm by RT‐PCR, suggesting that the genetic specification to cardiac lineage initiated in the mesoderm by 7.25 days p.c. The 7.25‐day p.c. isolated mesoderm in 48 hr culture, however, failed to differentiate into spontaneous beating cardiomyocytes and exhibited non‐organized MHC protein in 19% of these culture. In contrast, all of the 7.5‐day p.c. isolated mesoderm differentiated into beating cardiomyocytes even in 24 hr culture. The 7.25‐day p.c. mesoderm associated with primitive streak increased MHC protein expression in 93% of these cultures, although they formed beating foci in 3%. The 7.25‐day p.c. explants containing both visceral embryonic endoderm and primitive streak succeeded in terminal differentiation into spontaneous beating cardiomyocytes. Our study suggests that cardiac progenitor cells obtain the potency to complete terminal differentiation autonomously at 7.5 days p.c., as a consequence of the multistep induction by cell interactions with both the primitive streak and visceral embryonic endoderm, following the genetic specification to cardiac lineage in the early gastrula stage. Dev. Dyn. 1997;210:344–353.

Ionic basis of the chronotropic effect of acetylcholine on the rabbit sinoatrial node

OBJECTIVE The aim was to study the ionic basis of the chronotropic effects of bath applied acetylcholine and vagal stimulation on the rabbit sinoatrial node. METHODS The chronotropic effect of bath applied acetylcholine was measured in single cells and small multicellular preparations from the rabbit sinoatrial node and the chronotropic effect of postganglionic vagal stimulation was measured in the intact sinoatrial node. The roles of the hyperpolarisation activated current, i(f), the acetylcholine activated potassium current, iK,ACh, and the L-type calcium current, iCa, were investigated by blocking the currents with 1-2 mM Cs+ or 10(-6) M UL-FS49, 0.2-1.0 mM Ba2+, and 6 x 10(-6) M nifedipine, respectively. RESULTS Under control conditions, small multicellular preparations were approximately two orders of magnitude less sensitive to bath applied acetylcholine than single cells. However, after block of acetylcholinesterase by eserine in small multicellular preparations the sensitivities of the two types of preparation were approximately the same. Block of i(f) either had no discernible effect or increased the chronotropic effect of bath applied acetylcholine on single cells or small multicellular preparations, whereas partial block of iK,ACh reduced it substantially. Similarly, block of i(f) did not suppress the initial slowing of spontaneous action potentials by vagal stimulation, whereas partial block of iK,ACh reduced it. The hyperpolarisation of the arrested sinoatrial node in response to vagal stimulation was also substantially reduced by block of iK,ACh. Partial block of iCa caused large decreases in the action potential amplitude and maximum diastolic potential, but little decrease in the rate of spontaneous action potentials, and therefore did not mimic the effect of acetylcholine. CONCLUSIONS The chronotropic effects of bath applied acetylcholine and vagal stimulation are not principally the result of a suppression of i(f) or iCa, whereas the activation of iK,ACh may play an important role.

β-Adrenergic modulation of L-type Ca2+-channel currents in early-stage embryonic mouse heart

Little information is available concerning the modulation of cardiac function by β-adrenergic agonists in early-stage embryonic mammalian heart. We have examined the effects of isoproterenol (Iso) on the spontaneous beating rate and action potential (AP) configuration in embryonic mouse hearts at 9.5 days postcoitum (dpc), just 1 day after they started to beat. Iso (3 μM) increased the spontaneous beating rate in whole hearts, dissected ventricles, and isolated ventricular myocytes. In ventricular myocytes, Iso also increased the slope of the pacemaker potential and the action potential duration but decreased the maximum upstroke velocity. In whole cell voltage-clamp experiments, the Ca2+-channel currents were measured as Ba2+ currents ( I Ba). In 9.5-dpc myocytes, I Ba was enhanced significantly from -4.7 ± 0.9 to -6.7 ± 1.2 pA/pF (by 52.4 ± 14.8%, n = 10) after the application of Iso. Propranolol (3 μM) reversed the effect of Iso. Forskolin (For, 10 μM) produced an increase in I Ba by 95.5 ± 18.8% ( n = 8). In ventricular myocytes at a late embryonic stage (18 dpc), 3 μM Iso caused an appreciably greater increase in I Ba from -6.2 ± 0.5 to -14.5 ± 2.2 pA/pF (by 137.8 ± 33.0%, n = 8), whereas the increase in I Ba by 10 μM For (by 120.0 ± 23.0%, n = 7) was comparable to that observed in the early stage (9.5 dpc). These results indicate that the L-type Ca2+-channel currents are modulated by β-adrenergic receptors in the embryonic mouse heart as early as 9.5 dpc, probably via a cAMP-dependent pathway.