Disruption of gallbladder smooth muscle function is an early feature in the development of cholesterol gallstone disease

Abstract

BACKGROUND; Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis.

Methods: BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca(2+) transients were imaged in intact gallbladder.

Key results: Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist-induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca(2+) flashes, and their frequency is increased by carbachol (3 μm). After 1 week, lithogenic diet-fed mice exhibited disrupted Ca(2+) flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca(2+) stores, which are required for agonist-induced contraction, and diminished basal tone of the organ. Responsiveness of Ca(2+) transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4-8 weeks, concomitant with appearance of mucosal inflammatory changes.

Conclusions & inferences: These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.

Figure 1. Changes in biliary lipid compositions are observed in mice fed the lithogenic diet for only one week

Cholesterol saturation index (CSI) of bile (A) and hydrophobicity index (B) of bile salts are elevated in mice fed the lithogenic diet compared to mice fed the standard chow. CSI is corrected for urso- and muricholate because an increase in hydrophilic bile salts reduces micellar cholesterol solubility. C. Cholesterol crystals are present as early as one week, and most mice have crystals/stones by 2 weeks. D. An increase in the content of taurocholate (TC) and decrease in conjugated muricholate bile salts in LD fed-mice are responsible for the increased bile salt hydrophobicity since taurocholate (TC), taurochenodeoxycholate (TCDC) and taurodeoxycholate (TDC) (grayish shades) are more hydrophobic than tauro-alpha-muricholate (TaM), tauro-beta-muricholate (TbM), and tauroursodeoxycholate (TUDC) molecular species (bluish shades). Bile composition of mice fed a diet rich in cholic acid only resembled LD mice more closely while bile compositions of mice fed a diet enriched in cholesterol alone are similar to control mice (A, B and D). Neither the cholic acid- nor the cholesterol-enriched diets result in the presence of cholesterol crystals or stones in bile (C). Significantly different from control (chow) at P<0.05 (*), P<0.001 (***) and P<0.0001 (****) (ANOVA with Bonferroni's multiple comparison test).

Figure 2. Mice fed a lithogenic diet do not show inflammatory changes until 4 weeks on the diet

A. H & E stained sections of mouse gallbladder. Although subtle hyperplasia of the GBSM is observed in mice on the LD diet for 2 weeks, significant morphologic changes are seen only after 4 weeks on the LD. At 4 weeks, there is epithelial hyperplasia, more pronounced muscle hypertrophy and expansion of the lamina propria by inflammatory cells. At 8 weeks, the lamina propria continues to expand with mucosal folding into broad-based villi. There are inflammatory cells present in both the lamina propria and within the muscularis propria. In addition to hypertrophy, at 8 weeks the muscularis propria takes on a more disorganized architecture. B. Gallbladder sections immunostained with a monoclonal anti-mac-2 antiserum that detects macrophages and monocytes. Immunoreactive cells were sparse in mice fed chow or the LD for 1-2 weeks. At 4 weeks on the LD, a marked increase in the number of mac-2 positive cells is observed in the lamella propria, and extending into the muscularis propria.

Figure 3. Contractile responses of GBSM to cholinergic agonist are reduced in mice fed the LD for more than one week

A. Muscle tension recordings tension from mice fed a normal chow (upper trace) or LD (lower trace) for 8 weeks. The carbachol concentration was increased (0.1 to 10 μM) every 4 minutes as indicated beneath the traces. LD mice have increased spontaneous basal activity, indicated as small variations in tension before the addition of the agonist (arrow). B. Maximal carbachol responses obtained at doses ranging from 3-10 μM were reduced at 2 (*P<0.05), 4 (****P<0.0001) and 8 (**P<0.01) weeks on LD (n>8). C. Maximal responses to KCl (80 mM) were reduced only at 4 weeks (****P<0.0001) on LD (n>8). Contractility of GBSM in mice fed a cholic acid- or cholesterol-rich diet is comparable to control.

Figure 4. Disruption of synchronized and rhythmic activity in GBSM of mice fed a LD diet for 8 weeks

In control mice, rhythmic Ca²⁺ flashes are synchronized in a muscle bundle (traces from two different cells in one muscle bundle). After 8 weeks on the LD diet, Ca²⁺ flash activity is disorganized and three main patterns of Ca²⁺ activity are observed. Type I. Clusters of synchronized Ca²⁺ flashes interrupted by quiescent period (15-20 sec). Type II. Asynchronized Ca²⁺ flashes between GBSM cells of a single muscle bundle, with more or less rhythmic activity. Type III. Sporadic synchronized Ca²⁺ flashes with Ca²⁺ waves still active. Ca²⁺ flashes and Ca²⁺ waves throughout the recording period are indicated by orange and blue bars, respectively, beneath the traces. Disrupted flash activity would likely lead to decreased gallbladder tone and diminished intracellular Ca²⁺ stores.

Figure 5. Alterations in the occurrence of the various types of Ca²⁺ activities from 0-8 weeks on the LD

The three types of Ca²⁺ flash activity described for mice fed the LD for 8 weeks are found at earlier time points, and proportions vary with passage of time.

Figure 6. Time course of changes in basal and agonist-induced Ca²⁺ transient

A. In mice fed the standard chow, GBSM show rhythmic and synchronized Ca²⁺ flash activity as well as increased frequency of flashes in response to cholinergic activation. B. Basal activity was disrupted as early as the one-week time point, and at 4-8 weeks, carbachol failed to increase flash activity, but Ca²⁺ waves were observed.

Figure 7. Basal Ca²⁺ activity of GBSM in mice fed a cholic acid- or cholesterol-rich diet is comparable to mice fed a standard chow diet

In mice fed a diet enriched in cholic acid (upper trace) or cholesterol (lower trace) for 8 weeks, Ca²⁺ flashes are rhythmic and synchronized within a given bundle. In each set of traces, data from two GBSM cells from a single muscle bundle are illustrated. The orange bar under the trace represents Ca²⁺ flashes.