Altered ryanodine receptor gene expression in Hirschsprung's disease

Ryanodine receptors are the largest of all ion channels, named after their exogenous ligand, ryanodine. The ryanodine receptor calcium release channel is central to cytoplasmic Ca²⁺ signalling in skeletal muscle, the heart, and many other tissues, playing a vital role in muscular contraction. Three ryanodine receptors exist, Ryr1, Ryr2 and Ryr3. The ryanodine receptor, Ryr3, is encoded by the Ryr3 gene, which has been reported to be highly specific to colonic smooth muscle cells in mice. We designed this study to investigate Ryr1, Ryr2 and Ryr3 gene expression in the normal human colon and in Hirschsprung's disease (HSCR).

HSCR tissue specimens (n = 6) were collected at the time of pull-through surgery, while control samples were obtained at the time of colostomy closure in patients with imperforate anus (n = 6). qRT-PCR analysis was undertaken to quantify Ryr1, Ryr2 and Ryr3 gene expression, and immunolabelling of Ryr1, Ryr2 and Ryr3 proteins was visualised using confocal microscopy.

qRT-PCR analysis revealed a significant downregulation of the Ryr1 and Ryr3 genes in both aganglionic and ganglionic HSCR specimens compared to controls (p < 0.05). Confocal microscopy revealed Ryr1, Ryr2 and Ryr3 protein expressions within the smooth muscle, with a reduction in aganglionic and ganglionic HSCR colon compared to controls.

Ryr1 and Ryr3 gene expression is significantly downregulated in HSCR colon, suggesting a role for these genes in colonic smooth-muscle motility. Ryr1 and Ryr3 downregulations within ganglionic specimens highlight the physiologically abnormal nature of this segment which may explain the occurrence of persistent bowel symptoms in some HSCR patients following a properly performed pull-through operation.