Altered Penile Caveolin Expression in Diabetes: Potential Role in Erectile Dysfunction.

BACKGROUND: The pathophysiology of increased severity of erectile dysfunction in men with diabetes and their poor response to oral pharmacotherapy are unclear. Defective vascular endothelium and consequent impairment in the formation and action of nitric oxide (NO) are implicated as potential mechanisms. Endothelial NO synthase, critical for NO generation, is localized to caveolae, plasma membrane lipid rafts enriched in structural proteins, and caveolins. Type 2 diabetes mellitus (T2DM)-induced changes in caveolin expression are recognized to play a role in cardiovascular dysfunction. AIMS: To evaluate DM-related changes to male erectile tissue in a mouse model that closely resembles human T2DM and study the specific role of caveolins in penile blood flow and microvascular perfusion using mice lacking caveolin (Cav)-1 or Cav-3. METHODS: We used wild-type C57BL6 (control) and Cav-1 and Cav-3 knockout (KO) male mice. T2DM was induced by streptozotocin followed by a high-fat diet for 4 months. Penile expressions of Cav-1, Cav-3, and endothelial NO synthase were determined by western blot, and phosphodiesterase type 5 activity was measured using [3H] cyclic guanosine monophosphate as a substrate. For hemodynamic studies, Cav-1 and Cav-3 KO mice were anesthetized, and penile blood flow (peak systolic velocity and end-diastolic velocity; millimeters per second) was determined using a high-frequency and high-resolution digital imaging color Doppler system. Penile tissue microcirculatory blood perfusion (arbitrary perfusion units) was measured using a novel PeriCam PSI system. OUTCOMES: Penile erectile tissues were harvested for histologic studies to assess Cav-1, Cav-3, and endothelial NO synthase expression, phosphodiesterase type 5 activity, and blood flow, and perfusion measurements were assessed for hemodynamic studies before and after an intracavernosal injection of prostaglandin E1 (50 ng). RESULTS: In T2DM mice, decreased Cav-1 and Cav-3 penile protein expression and increased phosphodiesterase type 5 activity were observed. Decreased response to prostaglandin E1 in peak systolic velocity (33 ± 4 mm/s in Cav-1 KO mice vs 62 ± 5 mm/s in control mice) and perfusion (146 ± 12 AU in Cav-1 KO mice vs 256 ± 12 AU in control mice) was observed. Hemodynamic changes in Cav-3 KO mice were insignificant. CLINICAL TRANSLATION: Our findings provide novel mechanistic insights into erectile dysfunction severity and poor pharmacotherapy that could have potential application to patients with T2DM. STRENGTHS AND LIMITATIONS: Use of KO mice and novel hemodynamic techniques are the strengths. A limitation is the lack of direct evaluation of penile hemodynamics in T2DM mice. CONCLUSION: Altered penile Cav-1 expression in T2DM mice and impaired penile hemodynamics in Cav-1 KO mice suggests a regulatory role for Cav-1 in DM-related erectile dysfunction. Parikh J, Zemljic-Harpf A, Fu J, et al. Altered Penile Caveolin Expression in Diabetes: Potential Role in Erectile Dysfunction. J Sex Med 2017;14:1177-1186.

Age-related external anal sphincter muscle dysfunction and fibrosis: possible role of Wnt/ß-catenin signaling pathways.

Studies show an age-related increase in the prevalence of anal incontinence and sphincter muscle atrophy. The Wnt/ß-catenin signaling pathway has been recently recognized as the major molecular pathway involved in age-related skeletal muscle atrophy and fibrosis. The goals of our study were to 1) evaluate the impact of normal aging on external anal sphincter (EAS) muscle length-tension (L-T) function and morphology and 2) specifically examine the role of Wnt signaling pathways in anal sphincter muscle fibrosis. New Zealand White female rabbits [6 young (6 mo of age) and 6 old (36 mo of age)] were anesthetized, and anal canal pressure was measured to determine the L-T function of EAS. Animals were killed at the end of the study, and the anal canal was harvested and processed for histochemical studies (Masson trichrome stain for muscle/connective tissue) as well as for molecular markers for fibrosis and atrophy [collagen I, ß-catenin, transforming growth factor-ß (TGF-ß), atrogin-1, and muscle-specific RING finger protein-1 (MuRF-1)]. The L-T was significantly impaired in older animals compared with young animals. Anal canal sections stained with trichrome showed a significant decrease in the muscle content (52% in old compared with 70% in young) and an increase in the connective tissue/collagen content in the old animals. An increased protein and mRNA expression of all the fibrosis markers was seen in the older animals. Aging EAS muscle exhibits impairment of function and increase in connective tissue. Upregulation of atrophy and profibrogenic proteins with aging may be the reason for the age-related decrease in anal sphincter muscle thickness and function.NEW & NOTEWORTHY Our studies using a female rabbit model show age-related alterations in the structure and function of the external anal sphincter (EAS) muscle. We used endoluminal ultrasound to measure age-related changes in EAS muscle thickness. We employed Western blot and quantitative PCR to demonstrate age-related changes in the levels of important fibrogenic as well as atrophy markers. Our findings may have significant clinical implications, i.e., use of specific antagonists to prevent age-related EAS muscle dysfunction.

Wnt-ß Catenin Signaling Pathway: A Major Player in the Injury Induced Fibrosis and Dysfunction of the External Anal Sphincter.

Wnt-ß catenin is an important signaling pathway in the genesis of fibrosis in many organ systems. Our goal was to examine the role of Wnt pathway in the external anal sphincter (EAS) injury-related fibrosis and muscle dysfunction. New Zealand White female rabbits were subjected to surgical EAS myotomy and administered local injections of either a Wnt antagonist (sFRP-2; daily for 7 days) or saline. Anal canal pressure and EAS length-tension (L-T) were measured for 15 weeks after which the animals were sacrificed. Anal canal was harvested and processed for histochemical studies (Masson trichrome stain), molecular markers of fibrosis (collagen and transforming growth factor-ß) and immunostaining for ß catenin. Surgical myotomy of the EAS resulted in significant impairment in anal canal pressure and EAS muscle L-T function. Following myotomy, the EAS muscle was replaced with fibrous tissue. Immunostaining revealed ß catenin activation and molecular studies revealed 1.5-2 fold increase in the levels of markers of fibrosis. Local injection of sFRP-2 attenuated the ß catenin activation and fibrosis. EAS muscle content and function was significantly improved following sFRP-2 treatment. Our studies suggest that upregulation of Wnt signaling is an important molecular mechanism of injury related EAS muscle fibrosis and sphincter dysfunction.

Myoarchitectural and functional alterations in rabbit external anal sphincter muscle following experimental surgical trauma.

Obstetrical trauma to external anal sphincter (EAS) is extremely common; however, its role in the development of anal incontinence is not clear. We examined the regenerative process and functional impact of experimental surgical trauma to EAS muscle in an animal model. Surgical myotomy, a craniocaudal incision extending along the entire length and thickness of the EAS, was performed in rabbits. Animals were allowed to recover, and anal pressures were recorded at weekly intervals for 12 wk using a custom-designed probe system to determine the length-tension property of EAS muscle. Animals were killed at predetermined time intervals, and the anal canal was harvested for histochemical studies (for determination of muscle/connective tissue/collagen) and sarcomere length measurement. In addition, magnetic resonance diffusion tensor imaging (MR-DTI) and fiber tracking was performed to determine myoarchitectural changes in the EAS. Myotomy of the EAS muscle resulted in significant impairment of its length-tension property that showed only partial recovery during the 12-wk study period. Histology revealed marked increase in the fibrosis (connective tissue = 69% following myotomy vs. 28% in controls) at 3 wk, which persisted at 12 wk. Immunostaining studies confirmed deposition of collagen in the fibrotic tissue. There was no change in the sarcomere length following myotomy. MR-DTI studies revealed disorganized muscle fiber orientation in the regenerating muscle. We conclude that, following experimental injury, the EAS muscle heals with an increase in the collagen content and loss of normal myoarchitecture, which we suspect is the cause of impaired EAS function.

Anatomical disruption and length-tension dysfunction of anal sphincter complex muscles in women with fecal incontinence.

BACKGROUND: Anal sphincter complex muscles, the internal anal sphincter, external anal sphincter, and puborectalis muscles, play an important role in the anal continence mechanism. Patients with symptoms of fecal incontinence have weak anal sphincter complex muscles; however, their length-tension properties and relationship to anatomical disruption have never been studied. OBJECTIVE: This study aimed to assess the anatomy of the anal sphincter complex muscles with the use of a 3-dimensional ultrasound imaging system and to determine the relationship between the anatomical defects and the length-tension property of external anal sphincter and puborectalis muscles in women with incontinence symptoms and in control subjects. DESIGN: Severity of anal sphincter muscle damage was determined by static and dynamic 3-dimensional ultrasound imaging. The length-tension property was determined by anal and vaginal pressure with the use of custom-designed probes. PATIENTS: Forty-four asymptomatic controls and 24 incontinent patients participated in this study. MAIN OUTCOME MEASURES: The anatomical defects and length-tension dysfunction of anal sphincter complex muscles in patients with fecal incontinence were evaluated. RESULTS: The prevalence of injury to sphincter muscles is significantly greater in the incontinent patients than in the controls. Eighty-five percent of patients but only 9% controls reveal damage to ≥2 of the 3 muscles of the anal sphincter complex. Anal and vaginal squeeze pressures increased with the increase in the probe size (length-tension curve) in the majority of controls. In patients, the increase in anal and vaginal squeeze pressures was either significantly smaller than in controls or it decreased with the increasing probe size (abnormal length-tension). LIMITATIONS: We studied patients with severe symptoms. Whether our findings are applicable to patients with mild to moderate symptoms remains to be determined. CONCLUSIONS: The length-tension property of the external anal sphincter and puborectalis muscles is significantly impaired in incontinent patients. Our findings have therapeutic implications for the treatment of anal incontinence.

Role of puborectalis muscle in the genesis of urethral pressure.

PURPOSE: The internal (smooth muscle) and the external (rhabdosphincter striated muscle) urethral sphincters have important roles in the genesis of urethral closure pressure. The U-shaped pelvic floor puborectalis muscle is important in the closure of anal and vaginal orifices in humans. We defined the contribution of the puborectalis to urethral pressure. MATERIALS AND METHODS: A total of 11 female rabbits were anesthetized and prepared to measure urethral, vaginal and anal canal pressure using manometric methods. Pressure was recorded at rest, after administration of pharmacological agents and during electrical stimulation of the puborectalis and rhabdosphincter sphincter muscles. Phenylephrine, sodium nitroprusside (Sigma-Aldrich®) and rocuronium bromide (PharMEDium, Lake Forest, Illinois) were used to define the relative contribution of smooth and striated muscles to urethral pressure. Histology of the pelvic floor hiatus was also studied. RESULTS: At rest mean ± SEM maximum urethral pressure was 13 ± 6 mm Hg. Sodium nitroprusside (50 µg/kg) infusion resulted in a 30% to 40% decrease in resting urethral pressure (mean 7.2 ± 0.2 mm Hg). Phenylephrine produced a dose dependent increase in urethral pressure (mean 17 ± 6, 25 ± 5 and 29 ± 6 for 5, 10 and 50 µg/kg intravenously, respectively). Electrical stimulation of the puborectalis muscle induced a stimulus dependent increase in urethral, vaginal and anal canal pressure. On the other hand, rhabdosphincter stimulation induced a stimulus intensity dependent increase in urethral pressure only. The increase in urethral pressure after puborectalis muscle stimulation was more than twofold higher than after rhabdosphincter stimulation. CONCLUSIONS: Our data prove that the puborectalis, a component of the pelvic floor muscles, is an important contributor to urethral pressure in the rabbit.

Sustained improvement in the anal sphincter function following surgical plication of rabbit external anal sphincter muscle.

BACKGROUND: We recently found that the anal canal function and external anal sphincter contraction can be enhanced by surgically adjusting the EAS muscle sarcomere length in rabbits. A 20% length plication of the external anal sphincter muscle results in significant increase in the anal canal pressure and EAS muscle stress without affecting its passive tension. The durability of the beneficial effect of external anal sphincter muscle plication on the anal canal function is not known. OBJECTIVE: We studied the long-term effects of optimal length external anal sphincter plication on the anal canal pressure, external anal sphincter sarcomere length, and anal canal histology. DESIGN: Female rabbits (n = 16) were anesthetized and either sham (n = 4) or external anal sphincter plication (n = 12) surgery was performed. MAIN OUTCOME MEASURES: The effect of external anal sphincter plication on the anal canal pressure was determined every 2 weeks for 6 months in 6 animals. Anal canal was harvested for sarcomere length and histological assessment. RESULTS: External anal sphincter plication resulted in 50% to 60% increase in the anal canal pressure, and 80% to 90% increase in external anal sphincter muscle stress (during maximum electrical stimulus). The effect of plication was durable for the entire study period of 24 weeks. Sarcomere length increased from 2.11 ± 0.08 µm to 2.59 ± 0.03 µm immediately after plication and was 2.35 ± 0.08 µm at the end of 6 months. Histology revealed no significant differences in the muscle (30% vs 29%) or connective tissue components (70% vs 71%) of the anal canal between control and chronically plicated animals. CONCLUSIONS: Normal external anal sphincter muscle plication results in long-term enhancement of the anal canal function without any untoward effects on the tissue architecture in the rabbit. External anal sphincter muscle plication could be an important strategy to improve the anal canal function in patients with anal incontinence.