Cyclophosphamide-induced HCN1 channel upregulation in interstitial Cajal-like cells leads to bladder hyperactivity in mice

Abstract

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are confirmed to be expressed in bladder interstitial Cajal-like cells (ICC-LCs), but little is known about their possible role in cystitis-associated bladder dysfunction. The present study aimed to determine the functional role of HCN channels in regulating bladder function under inflammatory conditions. Sixty female wild-type C57BL/6J mice and sixty female HCN1-knockout mice were randomly assigned to experimental and control groups, respectively. Cyclophosphamide (CYP)-induced cystitis models were successfully established in these mice. CYP treatment significantly enhanced HCN channel protein expression and I_h density and significantly altered bladder HCN1 channel regulatory proteins. Carbachol (CCH) and forskolin (FSK) exerted significant effects on bladder ICC-LC [Ca²⁺]_i in CYP-treated wild-type (WT) mice, and HCN1 channel ablation significantly decreased the effects of CCH and FSK on bladder ICC-LC [Ca²⁺]_i in both naive and CYP-treated mice. CYP treatment significantly potentiated the spontaneous contractions and CCH (0.001-10 μM)-induced phasic contractions of detrusor strips, and HCN1 channel deletion significantly abated such effects. Finally, we demonstrated that the development of CYP-induced bladder overactivity was reversed in HCN1-/- mice. Taken together, our results suggest that CYP-induced enhancements of HCN1 channel expression and function in bladder ICC-LCs are essential for cystitis-associated bladder hyperactivity development, indicating that the HCN1 channel may be a novel therapeutic target for managing bladder hyperactivity.

Figure 1

Cyclophosphamide (CYP)-induced cystitis in WT and HCN1−/− mice. (a) Characteristic inflammation was detected in histological bladder sections of CYP-treated WT and HCN1−/− mice. Urothelial thickness (b) and bladder weight (c) were significantly increased in both CYP-treated WT and HCN1−/− mice (data represent the mean±s.d., N=10, ***P<0.001, versus naive mice). (d, f) IL-6 and TNF-α protein expression levels in the bladders of both WT and HCN1−/− mice were significantly elevated when these mice were treated with CYP (data represent the mean±s.d., N=6, ***P<0.001, versus naive mice). (e, g) IL-6 and TNF-α protein expression levels were significantly enhanced in the bladder samples of patients undergoing chemotherapy (data represent the mean±s.d., N=5, ***P<0.001, versus control sample).

Figure 2

Increased HCN channel protein expression levels and equal quantities of bladder ICC-LCs in CYP-induced cystitis. (a, c) The protein expression levels of the four HCN subtypes were increased in the CYP-treated group (data represent the mean±s.d., N=6, *P<0.05, **P<0.01 and ***P<0.001, versus naive mice). (b, d) The protein expression levels of the four HCN subtypes were significantly enhanced in the bladder samples of patients undergoing chemotherapy (data represent the mean±s.d., N=5, *P<0.05 and **P<0.01, versus control sample). (e) Immunofluorescence labeling of bladder ICC-LCs in naive or CYP-treated WT and HCN1−/− mice. C-kit (green)-positive ICC-LCs were visualized under 800 × magnification and ICC-LCs in white frames were showed under × 2400 magnification. (f) There was no significant difference in the number of bladder ICC-LCs between each group (data represent the mean±s.d., N=5, all P-values >0.05).

Figure 3

Changes in I_h properties in bladder ICC-LCs. (a) Characteristic traces of I_h currents in the bladder ICC-LCs of naive or CYP-treated WT and HCN1−/− mice evoked by a voltage-clamp protocol with a holding potential of −60 mV and a step potential ranging from −60 to −120 mV in decrements of 10 mV. Application of ZD7288 (50 μM) reduced I_h amplitudes in all four groups. (b) RT-PCR showed that only c-kit and GAPDH expression was visualized in the lanes, confirming the reliability of the isolated bladder ICC-LCs. (c) Fully activated current traces of I_h evoked at −120 mV, showing that CYP treatment significantly elevated I_h current amplitudes and HCN channel gating rates in WT and HCN1−/− mice, while HCN1 channel deletion significantly decreased I_h current amplitudes and HCN channel gating rates in CYP-treated and naive mice. Over a voltage range of −60 to −120 mV, (d, e) CYP-treatment significantly increased I_h current density in WT and HCN1−/− mice (data represent the mean±s.d., N=10, *P<0.05, **P<0.01 and ***P<0.001, versus naive mice), (f, g) while HCN1 channel knockout significantly decreased I_h density in CYP-treated and naive mice (data represent the mean±s.d., N=10, *P<0.05, **P<0.01 and ***P<0.001, versus WT mice).

Figure 4

Filamin A and NEDD4L protein expression levels are altered in cystitis. (a, c) Upon CYP treatment, Filamin A protein expression level in the bladder was significantly elevated, while NEDD4L protein expression level was significantly downregulated (data represent the mean±s.d., N=6, **P<0.01 and ***P<0.001, versus naive mice). (b, d) The protein expression levels of Filamin A and NEDD4L in the bladder samples of patients undergoing chemotherapy were significantly increased and decreased, respectively, compared with the protein expression levels of Filamin A and NEDD4L in the bladder samples of the control group (data represent the mean±s.d., N=5, *P<0.05 and **P<0.01, versus control sample).

Figure 5

Effects of FSK and CCH on intracellular calcium ion concentrations ([Ca²⁺]_i) in bladder ICC-LCs. (a, b) Six pictures from each group of naive or CYP-treated WT and HCN1−/− mice were chosen to represent the continuous changes in real-time [Ca²⁺]_i before and after drug (CCH, FSK, ZD7288) administration. CCH or FSK was applied after recording for 1 min, and ZD7288 was applied after recording for 2 min. (c, d) The effects of CCH and FSK on [Ca²⁺]_i in each group were expressed as relative fluorescence intensities (RFI, F1/F0), which were illustrated as continuous curves. Application of CCH (1 μM) and FSK (50 μM) significantly enhanced [Ca²⁺]_i in all four groups, while application of ZD7288 (50 μM) decreased [Ca²⁺]_i in all four groups. HCN1 channel deletion significantly weakened the effects of CCH (e) or FSK (f) on [Ca²⁺]_i in the bladder ICC-LCs of both naive and CYP-treated mice (data represent the mean±s.d., N=10, ***P<0.001, versus WT mice).

Figure 6

Altered detrusor strip spontaneous contractions and sensitivity to CCH. (a) Detrusor strips isolated from naive or CYP-treated WT and HCN1−/− mice exhibited different spontaneous contraction amplitudes (before CCH application) and varied responses to gradient doses of CCH (0.001–10 μM). (b, c) CYP treatment significantly potentiated the spontaneous contractions and CCH-induced phasic contractions of detrusor strips in WT and HCN1−/− mice (data represent the mean±s.d., N=8, **P<0.01 and ***P<0.001, versus naive mice). (d, e) HCN1 channel ablation significantly weakened the spontaneous contractions and CCH-induced phasic contractions of detrusor strips in both CYP-treated and naive mice (data represent the mean±s.d., N=8, **P<0.01 and ***P<0.001, versus WT mice).

Figure 7

The development of CYP-induced bladder overactivity was reversed in HCN1−/− mice. (a) Various cystometry patterns in naive or CYP-treated WT and HCN1−/− mice. CYP-induced obvious bladder overactivity in WT mice, which was reversed by HCN1 channel deletion. Absence of the HCN1 channel resulted in a relatively hypoactive bladder. Frequent NVCs (black arrow) were observed in both CYP-treated WT and HCN1−/− mice. HCN1 channel knockout significantly increased the ICI (b) and decreased BP (c), MBP (d) in both naive and CYP-treated mice (data represent the mean±s.d., N=8, **P<0.01 and ***P<0.001, versus WT mice).