Michael E. DiSanto

Enhanced Force Generation by Corpus Cavernosum Smooth Muscle in Rabbits With Partial Bladder Outlet Obstruction

PURPOSE Growing clinical evidence suggests that benign prostatic hyperplasia induced partial bladder outlet obstruction is associated with an increased incidence of erectile dysfunction. We determined whether corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction show any molecular or functional differences versus controls. MATERIALS AND METHODS Force generation and relaxation of corpus cavernosum smooth muscle 2 weeks after partial bladder outlet obstruction by 125 mM. KCl, phenylephrine and field stimulation were determined. Expression of total smooth muscle myosin and alternatively spliced smooth muscle myosin isoforms were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative competitive RT-PCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Corpus cavernosum smooth muscle from sections of the penis were analyzed morphologically by immunofluorescence microscopy using antibodies to smooth muscle myosin and neurofilament protein. RESULTS Corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction generated 40% to 50% more force than that of sham operated rabbits in response to KCl or phenylephrine and was more difficult to relax. Although quantitative competitive RT-PCR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that corpus cavernosum smooth muscle from rabbits with partial bladder outlet obstruction expressed only slightly more total smooth muscle myosin at the messenger RNA and protein levels, expression of the high adenosine triphosphatase isoform SM-B increased 2-fold. Morphological examination of corpus cavernosum smooth muscle sections revealed decreased innervation and increased smooth muscle bundle size. CONCLUSIONS We present the novel finding of molecular and functional changes in the corpus cavernosum smooth muscle associated with partial bladder outlet obstruction. Although the inclusion of sham operation ruled out direct injury during surgery, a change in corpus cavernosum smooth muscle innervation induced by nerve compression by the ligature is likely to induce trophic changes in the corpus cavernosum smooth muscle leading to over expression of the SM-B smooth muscle myosin isoform, increased contractility and impaired relaxation.

Expression of myosin isoforms in smooth muscle cells in the corpus cavernosum penis

Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses approximately 4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17 isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (approximately 10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches approximately 23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.Corpus cavernosum smooth muscle (CCSM) in the penis is unique in that it exhibits a high resting tone and, on stimulation, the muscle cells relax, allowing cavernous spaces to fill with blood, which results in an erection (tumescence). During detumescence, the muscle cells contract and return to the state of high resting tone. This study was undertaken to determine whether CCSM with these unique properties contains myosin isoforms typical of aorta or bladder smooth muscles, muscles that exhibit tonic and phasic characteristics, respectively. RT-PCR revealed that normal CCSM contains an SM2/SM1 mRNA ratio of 1.2:1 (similar to the rabbit aorta). Approximately 31% of the myosin heavy chain transcripts possess a 21-nt insert (predominant in bladder smooth muscle but not expressed in aorta) that encodes the seven-amino acid insert near the NH2-terminal ATP binding region in the head portion of the myosin molecule found in SMB, with the remaining mRNA being noninserted (SMA). Quantitative competitive RT-PCR revealed that the CCSM possesses ∼4.5-fold less SMB than the bladder smooth muscle. Western blot analysis using an antibody specific for the seven-amino acid insert reveals that both SM1 and SM2 in the CCSM contain the seven-amino acid insert. Furthermore, SMB containing the seven-amino acid insert was localized in the CCSM by immunofluorescence microscopy using this highly specific antibody. The analysis of the expression of LC17isoforms a and b in the CCSM revealed that it is similar to that of bladder smooth muscle. Thus the CCSM possesses an overall myosin isoform composition intermediate between aorta and bladder smooth muscles, which generally express tonic- and phasiclike characteristics, respectively. Two-dimensional gel electrophoresis showed a relatively low level (∼10%) of Ca2+-dependent light-chain (LC20) phosphorylation at the basal tone, which reaches ∼23% in response to maximal stimulation. The presence of noninserted and inserted myosin isoforms with low and high levels of actin-activated ATPase activities, respectively, in the CCSM may contribute to the ability of the CCSM to remain in a state of high resting tone and to relax rapidly for normal penile function.

Alteration of contractile and regulatory proteins following partial bladder outlet obstruction.

This paper reviews the contractility and the expression of contractile and regulatory proteins in the detrusor smooth muscle (DSM) following partial bladder outlet obstruction (PBOO) in rabbits. PBOO was surgically induced by partial ligation of the urethra in adult male New Zealand White rabbits. The force generated by DSM strips from normal and obstructed bladders which showed bladder dysfunction, despite detrusor hypertrophy (decompensated bladder, DB) was measured. The expression of contractile and regulatory proteins was analyzed by reverse transcriptase-polymerase chain reaction and Western blotting. The DSM from obstructed DB revealed an overexpression of SM-A myosin heavy chain isoform (associated with decreased maximum velocity of shortening). DSM from sham-operated rabbits showed phasic contractions, whereas the detrusor from DB was tonic, exhibiting slow development of force, a longer duration of force maintenance, and slow relaxation. Rho-kinase inhibitor Y-27632 enhanced the relaxation of precontracted (with 125 mM KCl) DSM strips from DB. The enhancement of relaxation of DB by Y-27632 was associated with dephosphorylation of myosin light chain. The detrusor from normal bladders expresses predominantly the smooth muscle caldesmon (h-CaD), a thin filament-associated protein. However, the DSM from DB shows an overexpression of l-CaD, the non-muscle isoform of CaD. The l-CaD colocalizes with myosin in the cytoplasmic filaments in myocytes. These results show that the alteration of contractility of the detrusor following PBOO is associated with changes in the expression of proteins that form the contractile apparatus and regulate the actomyosin ATPase activity and contraction.

Improved contractility of obstructed bladders after tadenan treatment is associated with reversal of altered myosin isoform expression

PURPOSE Tadenan is a plant extract from Pygeum africanum used in the treatment of benign prostatic hyperplasia, to protect the bladder from contractile dysfunction induced by partial bladder outlet obstruction (BOO). The aim of the present study was to determine whether the Tadenan-induced return of detrusor contractility affects the expression of myosin isoforms, which differ at the C-terminal (SM1 and SM2) and the N-terminal regions (SM-A and SM-B). MATERIALS AND METHODS Four groups of New Zealand White rabbits (3 to 5 kg., 4 to 6 rabbits per group) were either partially obstructed by ligation of the urethra (groups 1 and 2) or not obstructed (groups 3 and 4). After 2 weeks, rabbits from groups 2 and 4 received Tadenan in peanut oil (vehicle) orally at 100 mg. /kg./day for 3 weeks and rabbits in groups 1 and 3 received vehicle only. Rabbits were sacrificed and bladders were removed and weighed. Contractility studies were performed on isolated strips of detrusor and the remaining muscular layer from the bladder body was used to study the expression of myosin heavy chain (MHC) isoforms at mRNA (SM1, SM2, SM-A, and SM-B) and the protein (SM1 and SM2) levels by RT-PCR and SDS-PAGE analyses, respectively. RESULTS Tadenan significantly reduced the effect of BOO on bladder mass. The diminished contractile response to field stimulation and carbachol secondary to urethral obstruction was significantly reversed by Tadenan treatment. The relative ratios for MHC isoforms were altered at the mRNA (SM2:SM1 and SM-A:SM-B) and protein (SM2:SM1) levels in obstruction. Upon treatment with Tadenan, the ratio of these isoforms returned to normal, as shown at the mRNA levels. In addition, the altered relative ratio of SM2:SM1 at the protein level also returned to nearly normal values after treatment. CONCLUSIONS Improvement of obstruction-induced contractile dysfunction of the detrusor following treatment with Tadenan is associated with changes in the expression of myosin isoforms. The alteration in the expression of myosin isoforms associated with obstruction-induced hypertrophy is reversed close to normal in the detrusor smooth muscle from Tadenan-treated obstructed rabbits.

Contractile protein changes in urinary bladder smooth muscle following outlet obstruction.

Force production by the bladder body smooth muscle and relaxation of the bladder outlet, required for bladder emptying, are regulated by Ca2+ via myosin light chain (MLC) phosphorylation by a Ca2+-calmodulin-dependent MLC kinase (for review, 1–3). Besides this mode of regulation, evidence from most laboratories, including ours, supports the existence of a thin-filament-mediated regulation due to protein-protein interaction by actin-binding proteins acting in concert with tropomyosin and a calcium-binding protein, most likely, calmodulin. Caldesmon (CaD), a thin-filament-associated protein, inhibits the ac-tin-myosin interaction and actomyosin ATPase, and this inhibition is reversed by calmodulin in the presence of Ca2+.3–5 Urinary bladder outlet obstruction interferes with the ability of the bladder to empty its contents, thereby inducing changes in the bladder wall smooth muscle cells which enable the bladder to produce the increased contractile force required to expel urine through the obstructed urethra. In the initial phases of outlet obstruction, there is transient decompensation of the bladder smooth muscle, which initiates the molecular events that lead to hypertrophy of the detrusor smooth muscle cells enabling it to generate and maintain force. However, continuation of the outlet obstruction induces molecular, cellular, and structural alterations in the muscle cells of the bladder wall, leading ultimately to decreased compliance and impaired emptying. The ability of smooth muscles to compensate for increased functional demand is associated with alterations in the expression of proteins in the thick- and thin-filaments.

Longitudinal studies of time‐dependent changes in both bladder and erectile function after streptozotocin‐induced diabetes in Fischer 344 male rats

To provide sensitive physiological endpoints for the onset and long‐term progression of deficits induced by diabetes mellitus (DM) in bladder and erectile function in male rats, and to evaluate parallel changes in urogenital and nerve function induced by hyperglycaemia over a protracted period as a model for chronic deficits in patients with diabetes.

Partial Bladder Outlet Obstruction Induces Urethral Smooth Muscle Hypertrophy and Decreased Force Generation

PURPOSE PBOO leads to increased urinary frequency, decreased void volume, hypertrophy of the detrusor SM, and alterations in contractile and regulatory proteins. This study was done to determine whether PBOO induced increases in urinary frequency and detrusor SM hypertrophy are associated with an alteration in the contractility and expression of myosin isoforms in urethral SM. MATERIALS AND METHODS PBOO was surgically induced in male New Zealand White rabbits, and sham operated rabbits served as controls. After surgery, rabbits were kept 12 days, and prior to sacrifice, urine output and voiding frequency were monitored by keeping the animals in metabolic cages for 24 hours. Animals with increased urinary frequency (mean +/- SEM 43 +/- 12 voids per 24 hours) and sham operated rabbits (6 +/- 3 voids per 24 hours) were used for this study. Morphology of the urethra was studied using light and immunofluorescence microscopy. The expression of myosin isoforms was analyzed at the mRNA and protein levels by RT-PCR and Western blotting. RESULTS The urethral wall and SM of PBOO rabbits showed hypertrophy. The force produced by the longitudinal muscle strips of PBOO animals in response to phenylephrine, KCl, or electrical field stimulation was decreased 50%, 37% and 40%, respectively. Immunofluorescence microscopy revealed a decrease in nerve density. RT-PCR and Western blotting showed a decrease in the expression of myosin isoform SM-B with a concomitant increase in SM-A at the mRNA and protein levels. CONCLUSIONS Our data show hypertrophy of the urethral wall and SM, and alterations in contraction, innervation, and myosin isoforms in PBOO induced detrusor hypertrophy.

Changes in the composition of myosin isoforms in smooth muscle hypertrophy following urinary bladder outlet obstruction

Abstract The ability of smooth muscles to compensate for increased functional demand is associated with alterations in the expression and function of a number of contractile proteins and other proteins that are involved in excitation-contraction coupling and active force generation. However, continuation of the structural alterations in the muscle cells of the bladder wall leads ultimately to decreased compliance and impaired emptying. Decompensation of the bladder muscle with persistent outlet obstruction is likely to be caused by breakdown of the structure and function of proteins that form the contractile apparatus and those that enable smooth muscle cells to take up, store, and release Ca 2+ . This would affect the activation of the contractile apparatus. In this chapter, we review the contractile proteins that are important for force production and maintenance in smooth muscles and the effect of outlet obstruction on the expression of these proteins.

EFFECT OF CAVERNOUS NEUROTOMY ON CORPUS CAVERNOSUM SMOOTH MUSCLE: ALTERATION IN MYOSIN ISOFORM COMPOSITION AND THE SPHINGOSINE-1-PHOSPHATE/RHO-KINASE SIGNALING PATHWAY

INTRODUCTION AND OBJECTIVE: Erectile dysfunction (ED) is a significant problem for many men following radical prostatectomy surgery, even with significant advances in nerve-sparing techniques. Although much work has examined the effect of neurotomy on the nitric-oxide/cGMP and fibrosis signaling pathways, little focus has been placed on the corpus cavernosum smooth muscle (CCSM) contractile apparatus. We performed experimental unilateral (UCN) and bilateral (BCN) cavernous neurotomy for 2 & 4 weeks (wks) and subsequently examined the molecular and physiological changes in isolated corpus cavernosum (CC) tissue with specific emphasis on smooth muscle myosin (SMM) isoform composition and the sphingosine-1-phosphate (S1P)/Rho-kinase (ROK) signaling pathway. METHODS: Thirty male rats (275-300 g) were divided into 6 groups. Group 1 BCN for 2 wks, group 2 BCN for 4 wks, group 3 UCN for 2 wks, group 4 UCN for 4 wks, group 5 sham surgery for 2 wks, and group 6 sham surgery for 4 wks (n = 5 per group). CC tissue from each group was prepared for myographic organ bath physiology. In addition, SM myosin (SMM) isoform composition was determined by competitive RT-PCR, while expression of S1P/ROK signaling pathway molecules were determined by Real-Time RT-PCR. RESULTS: UCN induced significant increases in the relative expression of the SM2 & SM-B SMM heavy chain (4 wks) and LC17a essential SMM light chain (2 & 4 wks) alternatively spliced isoforms which are all associated with more phasic-type SM contractility. Expression of the ROK isoform, associated with increased spontaneous phasic contractions (SPCs) was increased (~ 2-fold) at 2 weeks in response to UCN. Sphingosine kinase-2 (SPHK2), that along with ROK is associated with increased apoptosis, was increased 29% and 64% at 2 & 4 wks post injury. Correlating with our molecular findings, organ bath physiology revealed increased SPCs in CCSM from both UCN and BCN rats. CONCLUSIONS: Our findings reveal a neurotomy-induced increase in ROK and a switch to a SMM isoform composition favoring a more phasic type contraction which is well supported by our physiological findings of increased SPCs. Increased expression of SPHK2 along with increased ROK also favors apoptosis. Taken together, our results suggest that molecular and physiological changes occurring at the level of the SM contractile apparatus may play a major role in nerve injuryinduced ED and may explain why erection does not always return even when nerve function is recovered.

MP58-12 A FOUR MIRNAS SIGNATURE AS A POTENTIAL BIOMARKER FOR PREDICTING SURVIVAL USING BIOINFORMATICS ANALYSIS IN BLADDER CANCER

Increasing evidences showed that a huge number of miRNAs were abnormally expressed in bladder cancer tissues and played vital roles in tumorigenesis, progression and metastasis. The aim of our study was to identify the differential miRNAs expression between bladder cancer and normal bladder tissues by analyzing the high-throughput miRNA data downloaded from TCGA database. Additionally, we evaluated the prognostic values of the differentially expressed miRNAs and constructed a four-miRNA signature that could effectively predict patient survival. According to the cut-off criteria (P<0.05 and |log2FC>1.2), a total of 348 differentially expressed miRNAs were identified between bladder cancer tissues and matched normal tissues, including 264 up-regulated miRNAs and 84 down-regulated miRNAs. The Kaplan-Meier survival method revealed the prognostic function of the four miRNAs (miRNA-217, miRNA-378c, miR-33b and miRNA-615). Univariate and multivariate Cox regression analysis showed that the four-miRNA signature was an independent prognostic factor in bladder cancer. The functional enrichment analysis showed that the target genes of four miRNAs may be involved in various pathways related to cancer, including Notch signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, cGMPPKG signaling pathway, FOXO and signaling pathway. Taken together, our study suggested that four-miRNA signature could be used as a prognostic marker in bladder cancer.