Homocysteine enhances cardiac neural crest cell attachment in vitro by increasing intracellular calcium levels

Abstract

Elevated homocysteine (Hcys) increases the risk of neurocristopathies. Previous studies show Hcys inhibits neural crest (NC) cell migration in vivo. However, the mechanisms responsible for this effect are unknown. Here, we evaluated the effect of Hcys on NC cell attachment in vitro and determined if any of the effects were due to altered Ca 2؉ signaling. We found Hcys enhanced NC cell attachment in a dose and substratedependent manner. Ionomycin mimicked the effect of Hcys while BAPTA-AM and 2-APB blocked the effect of Hcys on NC attachment. In contrast, inhibitors of plasma membrane Ca 2؉ channels had no effect on NC attachment. Hcys also increased the emission of the intracellular Ca 2؉ -sensitive probe, Fluo-4. These results show Hcys alters NC attachment by triggering an increase in intracellular Ca 2؉ possibly by generating inositol triphosphate. Hence, the teratogenic effect ascribed to Hcys may be due to perturbation of intracellular Ca 2؉ signaling.

Folate, Homocysteine and the Cardiac Neural Crest

“…The CHD that result from exposure to elevated homocysteine, especially those effecting the outflow tract, are likely to be related to the impact that homocysteine has upon the development of the cardiac neural crest, (Brauer and Rosenquist, 2002;Boot et al, 2003Boot et al, , 2004Boot et al, , 2006Tierney et al, 2004;Rosenquist et al, 2007;Heidenreich et al, 2008). A number of biological, chemical, and genetic mechanisms have been postulated for the teratogenic effect of homocysteine during heart development, some of which have been reviewed previously van Mil et al, 2010).…”

The N-methyl-d-aspartate receptor in heart development: A gene knockdown model using siRNA

“…Alternatively, it has been hypothesized that the increase in homocysteine that accompanies folate insufficiency is the actual risk factor. We [12-14] and others [15,16] have shown that hyperhomocysteinemia is associated with conotruncal defects in the chicken embryo model, while recent evidence indicates that maternal hyperhomocysteinemia also may be associated with conotruncal defects in humans [17]. In fact it appears that hyperhomocysteinemia, rather than hypomethylation, is a more important risk factor in the development of heart defects [18].…”

In vivo calcium dynamics during neural crest cell migration and patterning using GCaMP3

“…We have previously shown that early emerging trunk NC cells reach the DA in discrete migratory streams through the rostral somite halves, under the guidance of CXCR4/CXCL12 signaling (Kasemeier-Kulesa et al, 2010). We were surprised to find only random calcium transients during NC cell migration in the head and trunk, since previous in vitro studies have identified calcium activities in fibroblasts (Wei et al, 2010), NC cell delamination (Newgreen and Gooday, 1985) and attachment to the substrate (Heidenreich et al, 2008), and motor axon guidance (Hanson and Landmesser, 2004). In contrast, the pattern of calcium transients after NC cells reached the DA was exciting (Figs.…”

“…Typical calcium indicators, such as Fluo-4 AM (Heidenreich et al, 2008) require invasive delivery into embryos or can be soaked into cells and tissue in vitro by external application. This severely limits the ability to accurately deliver calcium indicators to discrete cell subpopulations within the developing embryo during precise times of complex morphogenetic events.…”