Stretch-Induced Down-Regulation of HCN2 Suppresses Contractile Activity

Abstract

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.

Keywords: hyperpolarization-activated and cyclic nucleotide-gated 2 channel; ileus; intestinal motility; mechanotransduction.

Figure 1

Changes in spontaneous contractile activity in response to HCN inhibition. (A) Spontaneous contractile activity (integral), (B) average contraction amplitude, and (C) tone (average minimum of the contractile cycle) in the ileum in response to ZD7288 (50 μM), (D) dose–response for the effects of HCN inhibition with ZD7288 on intestinal tone, (E) changes in calcium sensitivity in the ileum and duodenum in response to HCN inhibition with ZD7288. (n = 6 per group for Panels (A–C,E); n = 5 per group for Panel (D); Panels (A–D), *, p < 0.05 vs. DMSO only; **, p < 0.005 vs. DMSO only; ***, p < 0.0005 vs. DMSO only; Panel (E), *, p < 0.05 vs. DMSO at the same calcium concentration).

Figure 2

Changes in agonist-induced contractile activity. Tissue sections were treated with escalating doses of carbachol at 5 min intervals, and contractile activity was measured at each dose. (A) Maximum agonist-induced contractile activity in the duodenum and ileum after treatment with vehicle (DMSO) or ZD7288 to inhibit HCN2. (B) Dose–response for the effects of HCN inhibition with ZD7288 on maximum contractile activity. (C) The effects of HCN inhibition with zatebradine on agonist-induced intestinal contractile activity. (D) The effects of HCN inhibition with ZD7288 on agonist-induced intestinal contractile activity in the presence or absence of tetrodotoxin (TTX). (VEH is DMSO only; n = 6 per group for Panel (A), n = 5 for Panels (B,D), n = 8 per group for Panel (C); Panels (A,B): *, p < 0.05 vs. VEH, **, p < 0.005 vs. VEH; Panels (C,D): *, p < 0.05 vs. the respective vehicle at the same carbachol dose).

Figure 3

The impact of inflammation on HCN2 inhibition of intestinal contractile activity. THP1 cells were subjected to control or increased cyclical stretch for 4 h (see Methods for description of stretching protocols) and the “Condition media” were collected. Intestinal sections were treated with the media by replacing 10% of the organ chamber volume with the conditioned media. (A) The effects of conditioned media alone on spontaneous contractile activity (integral). (B) Changes in the inhibitory effects of HCN inhibition on spontaneous contractile activity in response to conditioned media. (C) Changes in the inhibitory effects of HCN inhibition on agonist-induced contractile activity in response to conditioned media. (VEH is DMSO only; n = 6 per group; Panel (A): *, p < 0.05 vs. CCS; Panel (B): *, p < 0.05 vs. VEH; **, p < 0.005 vs. VEH; Panel (C): *, p < 0.05 vs. the respective vehicle group for the same carbachol dose).

Figure 4

The impact of stretch on HCN2 inhibition of intestinal contractile activity. (A) Changes in the inhibitory effects of HCN inhibition on agonist-induced contractile activity in response to stretch. (B,C) The effects of increased stretch on smooth muscle tissue HCN2 mRNA (B) and protein (C) levels after applying 0.5 g (normal load), 1 g, and 2 g loads to intestinal tissue in the organ bath system. (VEH is DMSO only; Panel (A), n = 5 per group; Panels (B,C), n = 4–8 per group; Panel (A): *, p < 0.05 vs. DMSO at the same carbachol dose; Panels (B,C): *, p < 0.05 vs. 0.5 g load).

Figure 5

Changes in HCN2 mRNA and protein levels in response to increased cyclical stretch in primary human intestinal smooth muscle cells (hISMC) and THP1 cells (macrophage-like cell line). (A) HCN2 mRNA levels normalized to β-actin in hISMC and THP-1 cells after 4 h of controlled cyclical stretch (CCS) or increased cyclical stretch (ICS). (B,C) HCN2 protein levels in THP-1 (B) and hISMC (C) after 4 h of CCS or ICS normalized to GAPDH. (n = 7 and 5 per group in hISMC and THP-1 cells, respectively, in Panel (A); n = 6 per group in Panel (B); n = 3 per group in Panel (C); *, p < 0.05 vs. VEH; **, p < 0.005 vs. VEH).