TY - JOUR
AU - Smith, Phillip, P
AB - Abstract The Hyperpolarization activated, cyclic nucleotide gated (HCN) channel is a candidate mediator of neuroendocrine influence over detrusor tonus during filling. In other tissues, HCN loss with aging is linked to declines in rhythmicity and function. We hypothesized that HCN has an age-sensitive expression profile and functional role in adrenergic bladder relaxation. HCN was examined in bladders from young (2–6 months) and old (18–24 months) C57BL/6 female mice, using qRT-PCR, RNAScope, and Western blots. Isometric tension studies were conducted using bladder strips from young wild-type (YWT), old wild-type (OWT), and young HCN1 knock-out (YKO) female mice to test the role HCN in effects of β-adrenergic stimulation. Hcn1 is the dominant HCN isoform RNA in the mouse bladder wall, and is diminished with age. Location of Hcn RNA within the mouse bladder wall is isoform-specific, with HCN1 limited to the detrusor layer. Passively-tensioned YWT bladder strips are relaxed by isoproterenol in the presence of HCN function, where OWT strips are relaxed only in the presence of HCN blockade. HCN has an age-specific expression and function in adrenergic detrusor relaxation in mouse bladder strips. Bladder physiology, Detrusor, Aging Disorders of urinary control are a major contributor to impaired quality of life, expense, and morbidity in the elderly (1). In contrast to the suboptimal detrusor pressure-centered therapeutic paradigm, aging in humans and animal models is more consistently associated with disordered bladder volume sensitivity rather than degradation of detrusor capabilities (2). Furthermore, recent reports emphasizing CNS/ bladder interaction contribute to a new model of bladder control as an adaptive response to multiple physiologic challenges including bladder filling, mobility, cognitive decline, and social context and awareness (1). “Afferent noise,” postulated to ensure brain awareness of the bladder (3), is generated by uncoordinated spontaneous detrusor activity. Countering this pro-tension activity is autonomic sympathetic input to the detrusor, providing a neuroendocrine relaxing influence via the Gs-coupled β3 adrenoceptor (β3AR) (4). As sympathetic outflow is continuously variable and dependent upon afferent information about bladder volume, this brain-sympathetic-bladder-afferent-brain feedback loop is postulated as a modulator of afferent sensitivity to bladder content (5). Analogous centrifugal feedback exists in other sensory systems (6,7), and is characteristic of biologic adaptive mechanisms aimed at maintaining homeostasis in the face of physiologic stressors. Recently, the Hyperpolarization activated, cyclic nucleotide gated (HCN) ion channel, has been described in human and rat bladders (8,9). HCN and its associated current Ih contribute to the regulation of neuronal excitability and activity, cardiac pacing, and gut motility (10). Activation voltages and kinetic responsiveness of this tetrameric channel trend towards more positive voltages and more rapid kinetics in response to intracellular cAMP. Four isoforms have been identified, differing in their response to hyperpolarization, intracellular cAMP, and opening dynamics. Control sensitivity to cAMP links HCN to the β3AR mechanism following stimulation. Pharmacologic studies using rat bladder strips showed that blockade of HCN enhanced spontaneous detrusor activity and inhibited beta-adrenergic detrusor relaxation (11). Age-associated change in HCN isoform expression accompanied by functional decline has been described in cardiac and gonadal tissues (12–14). Loss of Ih current density in sinoatrial node contributes to loss of maximal heart rate and decreased exercise tolerance (15) and decreased responsiveness to pharmacologic modulation of heart rate (13). Age-associated loss of HCN expression in the bladder could contribute to age-associated declines in responses to sympathetic input, thereby constricting basal detrusor tonus regulation. If detrusor tonus regulation is an adaptive mechanism, limiting the response range diminishes homeostatic capacity, thereby increasing the risk of regulatory/adaptive failure. Based on the relevance of HCN to adrenergic detrusor relaxation and the impact of age on HCN expression in other systems, we hypothesized that expression of one or more HCN isoforms diminishes with age, and would be associated with altered detrusor responses to β3AR stimulation. Methods Animals Young (2–6 months old; Jackson Lab) and old (18–22 months old; NIA) C57BL/6J mice were used for our strip studies on wild-type mice (wild-type young [YWT], wild-type old [OWT]). Bladders from female HCN1 knock-out mice (B6 129S-Hcn1tm2Kndl/J, 2–3 months old, KO young [YKO]) were kindly donated by Dr. Arie Mobley, Western New England University, MA. Research was conducted under institutional IACUC-approved protocol, 101169. Animals were euthanized with CO2. Tissues were kept in iced bubbled buffer until use, in all cases within 90 minutes of harvest. Experiment methods are here summarized. Further details appear in the online supplemental material. RNA Quantitative real-time polymerase chain reaction (RT-qPCR) and RNAScope studies were conducted to discover relative quantities and location of HCN RNA. For RT-qPCR, pre-designed Biorad PrimePCR SYBR Green Assays for mouse Hcn1-4 (Unique Assay IDs: qMmuCID0015896 [Hcn1], qMmuCID0007901 [Hcn2], qMmuCID0022774 [Hcn3], and qMmuCID0022862 [Hcn4]) were used. Gene expression was calculated via a modified Pflaffl method utilizing two reference genes (Rps18 and B2m, found to be stably expressed across age in young and old bladders). Data were normalized to gene expression of young, sex-matched mice to give comparable fold changes by geometric averaging of multiple internal control genes (16). Statistical significance was determined using t-test on log10 transformed data, alpha = 5.000%. Manual chromogenic RNAscope was performed on sections from two young and two old female bladders by following protocols and using kit reagents and probes from the manufacturer (Advanced Cell Diagnostics, Newark, CA). 5-µm sections were prepared, and ACD pre-designed probes for Mm-Hcn1 and Mm-Hcn2 (Cat. 423651-C2 and 427001-C2) used, with a channel change to C1 for use with the RED kit. RNAscope Negative Control Probe (DapB, dihydrodipicolinate reductase [dapB] gene) and Positive Control Probe (Mm-Ppib, peptidylprolyl isomerase B gene) were also run (Cat. 310043 and 313911). Detection of specific probe binding sites was done with RNAscope 2.5 HD Reagent Kit-RED (ACD, Cat. 322350) as per manufacturer instructions. Hematoxalin/Eosin (HE) stains were also prepared from each bladder. Digital brightfield images at 4×, 20×, and 40× were recorded on a Nikon photomicroscope. Protein Bladders were harvested into cold PBS and blotted dry, weighed and placed in lysis buffer (33 µL/mg tissue, CellLytic MT, Millipore Sigma) containing protease inhibitors. Two young and two old female bladders were homogenized together for each sample. Membranes were incubated in 1:1,000 rat anti-HCN1 or 1:250 rat anti-HCN2 antibody in TBST+5% nonfat milk at 4°C (EMD Millipore, MAB5594, and MAB5596, respectively). Correct identification of HCN1 and HCN2 was ensured using a biotinylated ladder (Cell Signaling Technology). Ponceau S staining confirmed equal protein loading. β-actin antibody (Biolegend, 622102) was used to blot a second membrane containing the same samples or after stripping the HCN membranes. Membranes were exposed and imaged using a Bio-rad ChemiDoc MP Imager with signal accumulation. Function Only female mice were used in order to maintain sex consistency with the available YKO animals. Bladders were excised from mice just above the ureters, placed in iced bubbled buffer, and 1-mm transverse full-thickness bladder strips prepared. Strips were mounted between an anchor and an HSE-HA F30 force transducer (Harvard Apparatus) in a myobath continually perfused with a 37°C bubbled buffer. The strip was slowly tensioned and then allowed to equilibrate for at least 30 minutes prior to experimentation with a final stable tension of 0.8–1.0 gm. Preparatory experiments showed that despite continued slow declines in tension with time, after 30 minutes stabilization the decline was less than 1/10 of data variability. Additionally, mathematical correction was made for the decline to ensure effects were not overestimated. Data were acquired at 20Hz using Windaq Pro+ (DataQ, Dayton OH), analyzed using Microsoft Excel and compared with GraphPad software. Strips were exposed to sequential drug solutions in buffer, recording tension under each condition. Length was held constant for each strip. Tension data were recorded for at least 3 minutes after tension stabilization, and the data were acquired under four sequential conditions: Buffer-only (Buff), 1 µM Isoproterenol added to buffer (Buff+Iso), Buffer with an HCN blocking agent added, either 5 mM cesium chloride (CsCl) or 50 µM ZD-7288 (Buff+Block), and Buff+Block with 1 µM isoproterenol (Block+Iso). Following the initial isoproterenol test, the strip was washed with buffer, and re-contracted with 1 µM carbachol (CCh), then washed with buffer again prior to recording under Block condition. Finally, a CCh-induced contraction confirmed strip viability at the conclusion of the studies. Tension (time-domain) and spectral (frequency-domain) analyses were conducted. Data under each condition were transferred to linked Microsoft Excel workbooks to ensure analytic consistency among mouse groups and drug subgroups. Mean tension transitions from Buff to Buff+Iso, Buff to Buff+Block, and Block to Block+Iso were compared using repeated measures ANOVA with Tukey post hoc testing within each mouse group (YWT, YKO, OWT). To analyze low amplitude tension variations, spectral analysis used the 2,048 data points (103 seconds) immediately prior to isoproterenol exposure and immediately following tension stabilization after Buff+Iso and Block+Iso. Fast Fourier Transform (Excel) was applied and maximum magnitude in the spectrum 0.01–0.5 Hz determined. Mean values were calculated under each condition and compared within each age group using repeated measures ANOVA with Tukey post hoc test. Results Hcn1 is the Dominant HCN Isoform RNA in the Mouse Bladder Wall Hcn1 and Hcn2 were detectable by RT-qPCR in both young and old bladders in female mice (Cq < 33), whereas Hcn3 and Hcn4 were below detection levels under conditions used. Hcn1 expression decreases by 40% in the old mouse bladders (p = .01), whereas Hcn2 expression remained unchanged with age (Figure 1A). Hcn1 appeared consistently at a lower Cq than did Hcn2 suggesting Hcn1 is the dominant isoform in female WT mice (Figure 1A). The same was true for Hcn1 and Hcn2 expression in male mouse bladders (data not shown). Figure 1. View largeDownload slide (A) HCN mRNA bladder expression and age. Hcn-1 mRNA levels measured by RT-qPCR were approximately 40% lower in WT old female bladders when compared to WT young controls, with no apparent change in Hcn-2 expression. Data were normalized to gene expression of young mice to give comparable fold changes by geometric averaging of multiple internal control genes. Comparisons made by t-test on log10 transformed data, *p ≤ .05 (n = 3 per group). Hcn-1 is decreased in old compared to young WT female bladders (p = .008), whereas no changes in Hcn-2 were observed. Bars show means (SD). (B) HCN bladder protein expression and age. (B1) Western blot analysis confirms the presence of higher levels of HCN1 as opposed to HCN2 protein in the mouse bladder. HCN1 protein expression declines with age. (B2) Ponceau S staining of the PVDF membrane prior to staining with HCN1 or HCN2 antibodies verifies equal protein loading of the young and old samples. (B3) HCN1 KO mice are missing a band slightly above the 100kDa molecular weight marker, corresponding to mouse HCN1 in bladder (marked with an asterisk in YWT). YWT = young wild-type female bladder; YKO = young knockout female bladder; OWT = Old wild-type female bladder; RT-qPCR = quantitative real-time polymerase chain reaction; HCN = Hyperpolarization activated, cyclic nucleotide gated channel. Figure 1. View largeDownload slide (A) HCN mRNA bladder expression and age. Hcn-1 mRNA levels measured by RT-qPCR were approximately 40% lower in WT old female bladders when compared to WT young controls, with no apparent change in Hcn-2 expression. Data were normalized to gene expression of young mice to give comparable fold changes by geometric averaging of multiple internal control genes. Comparisons made by t-test on log10 transformed data, *p ≤ .05 (n = 3 per group). Hcn-1 is decreased in old compared to young WT female bladders (p = .008), whereas no changes in Hcn-2 were observed. Bars show means (SD). (B) HCN bladder protein expression and age. (B1) Western blot analysis confirms the presence of higher levels of HCN1 as opposed to HCN2 protein in the mouse bladder. HCN1 protein expression declines with age. (B2) Ponceau S staining of the PVDF membrane prior to staining with HCN1 or HCN2 antibodies verifies equal protein loading of the young and old samples. (B3) HCN1 KO mice are missing a band slightly above the 100kDa molecular weight marker, corresponding to mouse HCN1 in bladder (marked with an asterisk in YWT). YWT = young wild-type female bladder; YKO = young knockout female bladder; OWT = Old wild-type female bladder; RT-qPCR = quantitative real-time polymerase chain reaction; HCN = Hyperpolarization activated, cyclic nucleotide gated channel. Based on the RT-qPCR findings, search for HCN protein by Western Blot was limited to HCN1 and HCN2. HCN1 and HCN2 proteins were found to be present in mouse bladders, albeit at low concentrations (Figure 1B1). Due to the small number of samples and concerns regarding the linearity of densitometric analysis, formal comparisons were not made. HCN1 protein grossly appeared less dense in the old bladders; Ponceau staining shows equal loading (1/B2). YKO mice did not show HCN1 protein (Figure 1B3). Location of Hcn RNA Within the Mouse Bladder Wall is Isoform-Specific Hcn1 and Hcn2 were localized by RNAScope (Figure 2). Hcn1 was seen almost exclusively within the detrusor muscle layer in both age groups. In contrast, Hcn2 was found in the detrusor smooth muscle, lamina propria, and urothelium. No gross differences were seen in distribution between these layers. As Hcn3 and Hcn4 were below detection levels by PCR, RNAScope imaging was not directed against these isoforms. Figure 2. View largeDownload slide RNAScope to localize Hcn in bladder. Representative light microscopy images of RNAScope from two young wild-type (WT) female mice and two old WT female mice. Each dot is reflective of one RNA molecule. Panels A and B are probed for Hcn1 and Hcn2 mRNA. Panel A shows 20× images, showing differential distribution of Hcn1 mRNA to detrusor, and more diffuse but more sparse distribution of Hcn2 mRNA. Panel B shows 40× images providing further detail of detrusor and mucosal distributions of Hcn1 and Hcn2 mRNA. Panel C shows positive and negative control images. Online version in color. Figure 2. View largeDownload slide RNAScope to localize Hcn in bladder. Representative light microscopy images of RNAScope from two young wild-type (WT) female mice and two old WT female mice. Each dot is reflective of one RNA molecule. Panels A and B are probed for Hcn1 and Hcn2 mRNA. Panel A shows 20× images, showing differential distribution of Hcn1 mRNA to detrusor, and more diffuse but more sparse distribution of Hcn2 mRNA. Panel B shows 40× images providing further detail of detrusor and mucosal distributions of Hcn1 and Hcn2 mRNA. Panel C shows positive and negative control images. Online version in color. Mucosa-Intact Bladder Strip Tension Response to Isoproterenol Differs With Age and Presence or Absence of HCN Function In urothelium-intact transverse bladder strips under isometric conditions, bath/drug transitions in all three groups gave mixed results (Figure 3). In YWT bladders, 1 μM isoproterenol induced relaxation (−9.8% vs baseline) (3A) in the absence of HCN blockade and a loss of spectral magnitude (3B). In the presence of HCN blockade, no statistically significant change in either measure was induced by isoproterenol. In contrast, in OWT bladders 1 μM isoproterenol increased spectral magnitude but did not induce a significant change in tension. However, in the presence of HCN blockade, an 11.1% tension relaxation with decline of spectral magnitude was induced by isoproterenol. Isoproterenol had no significant effect on either measure in YKO strips. HCN blockers themselves had no significant tension or spectral effect in YWT, OWT, and YKO bladder strips. Comparing HCN blocker (CsCl vs ZD7288) showed no difference in effect between the two agents. Figure 3. View largeDownload slide (A) Tension analyses. Tension (gms) response to isoproterenol, without/with HCN block. YWT = Young WT, YKO = Young HCN1 KO, OWT = Old WT, n = 10 in each group. Buff = buffer only, Iso = isoproterenol 1 μM, Block = buffer with added HCN blocker; HCN = Hyperpolarization activated, cyclic nucleotide gated channel. For each mouse type, HCN blockade with 5 mM CsCl (n = 5), 50 μM ZD7288 (n = 5). Comparisons by repeated measures ANOVA with Tukey post hoc test, * signifies p < .001. The net effect of loss of HCN1 function in young female mouse bladders is diminished relaxation to beta-adrenergic agonist, whereas in old bladders HCN blockade enhances response to isoproterenol. (B) Power Spectral Analyses, 0.01–0.5 Hz of tension data presented in Figure 3. Maximum spectral magnitude (logarithm to normalize distribution), taken from the final 2,048 data points in Buffer/Block and first 2,048 data points (20 Hz sampling) after stabilization following addition of isoproterenol. B-Iso = Buffer with block + 1 μM isoproterenol. For each mouse type, HCN blockade with 5 mM CsCl (n = 5), 50 μM ZD7288 (n = 5). Comparisons by repeated measures ANOVA with Tukey post hoc test. Isoproterenol suppresses spontaneous activity (as measured by magnitude) in YWT bladder strips in the absence of HCN blocker; however, YKO shows no impact. OWT bladders respond with enhanced magnitude in the absence of HCN block, and diminished magnitude after HCN blockade. Figure 3. View largeDownload slide (A) Tension analyses. Tension (gms) response to isoproterenol, without/with HCN block. YWT = Young WT, YKO = Young HCN1 KO, OWT = Old WT, n = 10 in each group. Buff = buffer only, Iso = isoproterenol 1 μM, Block = buffer with added HCN blocker; HCN = Hyperpolarization activated, cyclic nucleotide gated channel. For each mouse type, HCN blockade with 5 mM CsCl (n = 5), 50 μM ZD7288 (n = 5). Comparisons by repeated measures ANOVA with Tukey post hoc test, * signifies p < .001. The net effect of loss of HCN1 function in young female mouse bladders is diminished relaxation to beta-adrenergic agonist, whereas in old bladders HCN blockade enhances response to isoproterenol. (B) Power Spectral Analyses, 0.01–0.5 Hz of tension data presented in Figure 3. Maximum spectral magnitude (logarithm to normalize distribution), taken from the final 2,048 data points in Buffer/Block and first 2,048 data points (20 Hz sampling) after stabilization following addition of isoproterenol. B-Iso = Buffer with block + 1 μM isoproterenol. For each mouse type, HCN blockade with 5 mM CsCl (n = 5), 50 μM ZD7288 (n = 5). Comparisons by repeated measures ANOVA with Tukey post hoc test. Isoproterenol suppresses spontaneous activity (as measured by magnitude) in YWT bladder strips in the absence of HCN blocker; however, YKO shows no impact. OWT bladders respond with enhanced magnitude in the absence of HCN block, and diminished magnitude after HCN blockade. Discussion Our results suggest HCN is an age-sensitive mediator of neuroendocrine regulation over detrusor tonus during bladder filling, and therefore as a feature of adaptive regulation of bladder volume sensitivity. First, we confirm prior findings (11) that HCN is present and operational in adrenergic relaxation of detrusor, and second we show that the decline in HCN1 isoform with aging is associated with a markedly different response to adrenergic-induced detrusor relaxation. The location and mechanism of action of HCN in these responses remain to be determined, but our data provide some clues to guide future investigation. Our bladder strip analysis utilized both time-domain (tension) and frequency-domain (spectral power) analysis to extract maximum interpretable data from our experiments. Tension in a strip is the summation of passive (extracellular matrix) and active (myocyte/muscle) activity (17); small quasi-periodic variations in tension (micromotions, low amplitude rhythmic contractions) provide insight into myocyte activity and its coordination as it relates to overall strip tension (18). In both YWT (in buffer) and OWT (following HCN block), detrusor relaxation was accompanied by loss of spectral magnitude, suggesting that regional coordination of myocyte activity rather than diffuse uncoordinated activity drives tensions. The failure of isoproterenol to induce relaxation yet increase spectral magnitude in OWT in the absence of HCN block, suggests that adrenergic-induced changes in Ih dynamics in old bladders might augment autonomous activity by enhancing a primary pacemaker role of HCN. Since norepinephrine at low concentration can stimulate detrusor tension, our observations could reflect either an HCN-mediated change in the mechanics of adrenoreceptor response or a shift in the isoproterenol dose-response curve. Limitations of this study include: A full four–age group study (Young/Mature/Old/Oldest Old) is needed to understand the relative impacts of maturation versus aging on these mechanisms (19). Next, while historically regarded as standard HCN blocking agents, CsCl, and ZD-7288 are not highly specific for HCN blockade. In both cases, drug concentrations were chosen based on prior reports using these agents for HCN block in bladder or gut (8,11,20–23). Two agents with differing contaminating effects were chosen with the thought that similar responses in our experiments, as we found, would provide reassurance that observed effects were HCN-mediated. The potential for contaminating influence, however, remains a concern and will be resolved in future experiments supported by this study. Finally, our YKO mice were not of the same background as the WT mice. As this study was a proof-of-concept, we made use of available similar animals. While strain variances in urinary performance in colony mice have been shown, the similarity of effect among our mouse and reported rat bladder tissue responses (11) to HCN modulation suggest that genetic variance between our WT and KO was not a major determinant of our results. Our current data suggest that loss of dominant HCN1 function with aging contributes to degraded responses to autonomic stimulation, resulting in loss of adaptive control precision over detrusor tone—and therefore afferent responses to bladder volume—during filling. This diminished range of opportunity for adaptive success contributes to an increased risk of dysfunction without excluding normal function. Funding This work was supported by National Institutes of Health / National Institute on Aging K76 AG054777-01 Beeson Emerging Leaders Career Development Award in Aging (PI: P.P.S.); University of Connecticut Institute for Brain and Cognitive Science Seed Grant (PI: P.P.S./D.K.M.). Acknowledgments The authors wish to thank Arie Mobley PhD, University of Western New England, for her kind access to HCN1 KO mice. Conflict of Interest None of the authors report financial or academic conflicts of interest relating to this work. References 1. Vaughan CP , Markland AD , Smith PP , Burgio KL , Kuchel GA , the American Geriatrics Society/National Institute on Aging Urinary Incontinence Conference Planning Committee and Faculty . Report and research agenda of the American geriatrics society and national institute on aging bedside-to-bench conference on urinary incontinence in older adults: a translational research agenda for a complex geriatric syndrome . 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TI - HCN as a Mediator of Urinary Homeostasis: Age-Associated Changes in Expression and Function in Adrenergic Detrusor Relaxation
JF - The Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences
DO - 10.1093/gerona/gly137
DA - 2019-02-15
UR - https://www.deepdyve.com/lp/oxford-university-press/hcn-as-a-mediator-of-urinary-homeostasis-age-associated-changes-in-c7HtfANkpd
SP - 325
VL - 74
IS - 3
DP - DeepDyve
ER -