Comparisons of different stages of chick embryonic development by the physiological regulatory response to hyposmotic challenge

Abstract

Cardiac myocytes isolated and cultured from 11 day chick embryos present a Ca2+-dependent regulatory volume decrease (RVD) when exposed to hyposmotic stimulus. The RVD of myocytes from different embryonic stages were analyzed to evaluate their physiological performance through development. Among the several embryonic stages analyzed (6, 11, 16 and 19 days) only 19 day cardiac myocytes present a greater RVD when compared with 11 day (considered as control), the other ages showed no difference in the regulatory response. As it is known that RVD is Ca2+ dependent, we decided to investigate the transient free Ca2+ response during the hyposmotic swelling of the 11 and 19 day stages. The 11 day cardiac myocyte showed a transient 40% increase in intracellular free Ca2+ when submitted to hyposmotic solutions, and the free Ca2+ returned to baseline levels while the cells remained in hyposmotic buffer. However, the intracellular free Ca2+ transient in the 19 day cells during hyposmotic challenge increases 100% and instead of returning to baseline levels, declines to 55% above control, well after the 11 day transient has returned to baseline. Also, quantitative fluorescence microscopy revealed that 19 day cardiac myocytes have more sarcoplasmic reticulum (SR) Ca2+ ATPase sites per cell as compared to the 11 day cells. Our findings suggest that 19 day cells have more developed intracellular Ca2+ stores (SR). By evoking the mechanism of Ca2+ induced Ca2+ release, the cells have more free Ca2+ available for signaling the RVD during hyposmotic swelling.