To identify differential protein expression in penile tissue in a rat model of erectile dysfunction (ED) at an early stage after bilateral cavernosal nerve (CN) neurectomy, using proteomic techniques.
To identify differential protein expression in penile tissue in a rat model of erectile dysfunction (ED) at an early stage after bilateral cavernosal nerve (CN) neurectomy, using proteomic techniques.
Twelve male adult Sprague-Dawley rats were randomly divided into two equal groups, one having bilateral CN resection and one a control group. The penises were harvested 7 days after CN resection. Total protein was separated into >1250 protein spots by two-dimensional electrophoresis using pH 3–10 nonlinear immobilized pH gradient strips. Differential expression of proteins was analysed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database searching.
Thirty-two proteins were significantly changed in the denervated penis, of which 25 (including nine up-regulated and 16 down-regulated) with cytoskeletal functions, and pathophysiological functions related to energy metabolism and oxidative stress, were identified. Examples include transgelin, creatine kinase B, annexin-1 and galactin-7.
The expression of several important proteins participating in pathophysiological processes of penile tissue are changed early after bilateral CN neurectomy. These changes might give new insights into the cellular and molecular mechanisms involved in neurogenic ED development, and indicate potential therapeutic targets.
immobilized pH gradient
matrix-assisted laser desorption/ionization time-of-flight mass spectrometer
Coomassie Brilliant Blue
Erectile dysfunction (ED) remains one of the major problems in patients after radical prostatectomy (RP), despite major advances in operative technique [1,2]. It is generally thought that neurogenic ED is produced because of injury to the cavernosal nerve (CN) . Other investigators think a vascular factor is responsible, because evidence of hypoxia has been found after RP in the corpus cavernosum [3,4]. There was also apoptosis of corpus cavernosum smooth muscle cells at an early stage in an animal model of CN neurectomy [5,6]. Long-term CN neurectomy leads to fibrosis of the corpus cavernosum and increased TGF-β levels, which might contribute to this change in penile structure [4,7]. However, the mechanism of the structural damage remains to be elucidated. Investigators have used various methods to treat this nerve damage and reported an improvement in erectile function in an animal model . These improvements are usually associated with the decrease or increase of certain proteins in the corpus cavernosum.
Because the pathophysiology of CN neurectomy is not well understood, knowing the molecular mechanism of changes in penile tissue after CN neurectomy becomes more important for treating neurogenic ED. Proteomics is a useful tool for generating overall profiles of complex protein mixtures in pathological tissue . To elucidate the molecular mechanism of neurogenic ED at an early stage, we used a proteomic method to identify early protein biomarkers of damage, and thus potential therapeutic targets, in rat penis after CN denervation (a model mimicking the situation in patients who have had non-nerve sparing RP).
Twelve male adult Sprague-Dawley rats (3 months old, 300–350 g body weight) were randomly divided into two equal groups, one having bilateral CN resection (denervated) and a control group. The procedures were performed under aseptic conditions. Before surgery, all rats received general anaesthesia by an i.p. injection with 30 mg/kg of sodium pentobarbital. Through a lower midline incision, the bladder and major pelvic ganglion were located with a surgical microscope. After removing the fascia and fat from the dorsolateral lobe of the prostate, the afferent and efferent branches of the major pelvic ganglion were identified, and identification of the CN confirmed by inducing penile erection through electrostimulation. In the CN resection group, a 5-mm segment of the CN was removed bilaterally; small ancillary branches of the CN were also cut to prevent regeneration of the CN. In the control group, the bilateral CNs were identified but not cut. After surgery all rats were housed with a ‘12-h on/12-h off’ light cycle and allowed free access to water and food. Seven days later, the penile tissue was harvested, washed free of blood with normal saline, and tissue samples were snap-frozen in liquid nitrogen. All experimental procedures were conducted in accordance with standard guidelines for animal experiments and received approval from the Animal Care and Use Committee of the National Animal Ethics Commission.
The glans and fascia of penile tissue were removed and the trunk of penis was ground into powder in liquid nitrogen, homogenized in lysis buffer comprising 7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), 0.5% dithiothreitol (DTT), 0.25% Pharmalyte and 1 mm phenylmethylsulphonylfluoride, and sonicated at 40% maximum intensity (0.8 s, 10 times). The resulting homogenate was centrifuged at 20 000 g for 1 h at 4°C. The supernatant was collected, assayed to determine its total protein concentration using the a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), divided into aliquots, and stored at −80 °C.
Samples were assessed by first-dimension isoelectric focusing (IEF) on the Protean IEF Cell apparatus (Bio-Rad) at 20 °C. Each sample extract (300 µg of protein per sample) was first separated on an 18-cm nonlinear gradient immobilized pH gradient (IPG) strip, pH 3–10 (Amersham-Pharmacia, Sweden), and then silver-stained. These dry strips were actively rehydrated for 12 h at 50 V in rehydration buffer (7 m urea, 2 m thiourea, 4% CHAPS, and 65 mm DTT, all Bio-Rad, 0.2% w/v, Pharmalyte and a trace of bromophenol blue) containing total protein. The amount of total protein per sample added per gel was 300 µg for silver staining and 1 mg for modified Coomassie Brilliant Blue (CBB) G250 staining. The linear ramping mode of IEF voltage was used as follows: 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and 5000 V for 3 h, followed by 10 000 V to achieve 60 kVh. After IEF, the strips were stored at −20 °C until second-dimension electrophoresis.
After IEF, for the second-dimension separation we used a Bio-Rad Multicell system. The IPG strips were equilibrated in equilibration buffer (6 m urea, 2% SDS, 0.375 m Tris-HCl, pH 8.8, and 20% glycerol) at room temperature for 15 min. In the first and the second step, 2% DTT or 2.5% iodoacetamide were added to the equilibration buffer, respectively. In the second-dimension electrophoresis, the IPG strips were loaded onto a 12% SDS-PAGE gel. Running buffer (25 mm Tris, 192 mm glycine, and 3.5 mm SDS, pH 8.3) was added, and a constant current (5 W/gel) was applied for 6 h. The gels were stained with blue silver stain CBB G250  (Bio-Rad) or silver stain . The gels were scanned using a GS-800 Calibration Densitometer (Bio-Rad). PDQuest software (Bio-Rad) was used for spot detection, quantification and statistical analysis. The matching errors were evaluated manually. To determine spot molecular weight and pI, a two-dimensional marker was used (Bio-Rad). The CBB stain gel and the silver stain gel were analysed separately. Two separate gels were analysed, yielding six samples in each group. To minimize the experimental variation, the intensity of each spot was normalized against the sum total of intensities of all valid spots on the gel. The protein spots with significant changes (P < 0.05) in a consistent direction (increase or decrease) in all samples of the same group were considered to be different, and selected for further analysis and identification.
The protein spots identified as significant by change in density (P < 0.05) were manually excised from the two-dimensional gels with a clean scalpel and minced into 1-mm3 pieces, transferred into a clean 1.5-mL microcentrifuge tube, washed with a solution containing 50% acetonitrile and 50 mm ammonium bicarbonate, and then incubated at room temperature for 15 min (at least twice) until the colour of the gel disappeared. Complete dehydration was achieved by incubation of gel slabs with 100% acetonitrile, and then the gel pieces were reduced by placing them in mixture of 10 mm DTT in 100 mm ammonium bicarbonate for 1 h at 56 °C. After replacing DTT with a 55-mm iodoacetamide solution, the pieces were placed in the dark for 30 min, and then rehydrated (37 °C, overnight) in trypsin solution (10 mg/mL in 25 mm NH4HCO3; Promega, Madison, WI, USA). Peptides were extracted from the gel pieces with 60% acetonitrile in 1% trifluoracetic acid, and the extracts were dehydrated and then mixed with matrix, transferred to target plates, and allowed to dry. An Ettan matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (MS) was used to analyse the dried peptide-matrix spots.
Mass spectra of the peptides were acquired and compared to spectra generated from amino-acid sequences drawn from the NCBInr protein sequence database using the Mascot (http://www.matrixscience.com/home.html) search engine. The rat subdatabase was used and one missing cleavage point of trypsin was added to the database searching. The search variables used were: rat, trypsin digest, carbamidomethyl modification and peptide tolerance of 100 parts per million.
Proteins were well separated on pH 3–10 nonlinear but not linear IPG strips (data not shown). Using PDQuest software analysis, a mean (sd) of 1256 (84) protein spots were detected from the gel image. Most of these protein spots were at pH 4–9. The protein expression maps of control (left) and denervated (right) rat penis are shown in Fig. 1; among these, 841 were matched (match ratio ≈67%). The intensity of 32 spots was significantly changed by denervation; these spots were cut and identified by MALDI-TOF MS combined with database searching. Twenty-five of these spots were successfully identified. Other proteins were not identified successfully because of their low levels of expression. Of the 25 proteins identified, 12 were up-regulated and 13 were down-regulated (Table 1). Most of the identified proteins were involved in energy metabolism, oxidative stress and smooth muscle structure, e.g. spot 18 (which was significantly down-regulated after denervation) was identified as transgelin (SM22), a specific smooth-muscle protein (Fig. 2).
|Protein number/name||NCBInr accession no.*||Theoretical Mr/pI, kDa||Observed Mr/pI, kDa||Peptide coverage (%)||Change ratio†|
|Energy and metabolism|
|1 Creatine kinase B||gi|203476||42.9/5.4||48.3/5.4||29||−2.15|
|3 Pyruvate dehydrogenase (lipoamide) β||gi|50925725||38.8/5.9||42.6/5.8||52||2.42|
|4 ATP synthase β subunit||gi|1374715||51.1/4.9||50.4/4.8||32||1.27|
|5 Mitochondrial aldehyde dehydrogenase precursor||gi|45737864||56.07/6.4||53.3/6.5||25||−1.58|
|6 Enolase 3, β||gi|54035288||46.9/7.0||41.1/7.7||17||1.92|
|7 Haemoglobin α chain||gi|1304381||15.2/8.4||15.8/7.8||26||16.68|
|8 0β-1 globin||gi|818015||16.0/7.9||15.8/8.0||16||6.43|
|9 Haemoglobin β chain, minor||gi|92362||15.9/9.0||15.8/9.0||35||4.63|
|Oxidation and oxidative stress|
|10 NADH dehydrogenase 1α subcomplex 10 (ubiquinone)||gi|46391108||31.9/5.4||32.1/5.6||39||−1.60|
|11 Aldehyde reductase 1||gi|6978491||36.7/6.8||33.5/6.7||24||1.53|
|12 Peroxiredoxin 2; thioredoxin||gi|8394432||21.7/5.3||27.9/5.2||27||6.80|
|13 Cu/Zn SOD||gi|1213217||16.0/5.7||16.3/6.1||17||2.17|
|14 Myosin light chain 1 (MLC 1F)||gi|127131||20.6/5.0||25.0/4.9||44||−11.30|
|15 Tropomyosin 1α chain||gi|92090646||32.6/4.6||32.7/4.7||23||−5.52|
|16 Similar to actin, cytoplasmic 2 (γ-actin)||gi|109492380||62.5/7.0||61.3/7.0||21||2.34|
|17 Transgelin (SM22α)||gi|38197668||22.5/8.8||21.5/7.4||31||1.75|
|18 Transgelin (SM22α)||gi|6755714||22.5/8.8||19.2/7.6||22||3.31|
|19 Similar to myosin regulatory light chain 2||gi|109469114||19.7/4.8||17.9/4.8||26||−4.02|
|23 Apolipoprotein A-I||gi|2145147||29.9/5.5||25.0/5.2||22||1.87|
|24 Apolipoprotein A-I||gi|2145143||29.8/5.5||27.2/5.4||29||1.95|
|25 Fibrinogen β polypeptide||gi|29789106||54.2/7.9||49.5/7.3||17||−1.52|
It is generally thought that ED after RP is primarily caused by CN injury during surgery; unfortunately, almost all patients have ED immediately, even with bilateral nerve-sparing techniques, and delayed recovery or loss of erectile function remains an obstacle to their quality of life [1,2]. Although the high rate of ED after RP is well recognized, the cause and molecular pathophysiological mechanism have not been fully elucidated. Irreversible damage to the penis has been detected after surgery in humans and in animal models [4,12]. In the present study we found previously unreported changes in proteins in the denervated penis early after surgery, using a proteomic method.
The identification of protein changes specific to diseases using proteomic technology has the potential to clarify pathophysiological mechanisms involved in ED after surgery, thus assisting in selecting treatment. Many gene alterations were recently reported in the denervated penis using a microarray method , and to our knowledge the present is the first time that the overall profile of proteins in the penis has been reported. Most of the proteins for which the expression was significantly altered are critical to cellular energy metabolism and oxygen supply (36%). The expressions of the ATP synthase β subunit (spot 4), pyruvate dehydrogenase β (spot 3), α-enolase (spot 2), and enolase 3β (spot 6), enzymes critical in energy metabolism, were significantly down-regulated in the denervated penis. Similar results were found in atrophic skeletal muscle . Enolase, one of the enzymes involved in the energy-yielding phase of glycolysis, functions to convert the compound 2-phosphoglycerate to phosphoenolpyruvate. Its down-regulation is probably the result of hypoxia induced by CN resection , and leads to dysfunction of energy metabolism in the denervated corpus cavernosum. This phenomenon is further supported by the significant down-regulation of several haemoglobin proteins, including haemoglobin α (spot 7), 0β-1 globin (spot 8), and β chain (spot 9), which might affect the transfer of oxygen from erythrocytes to corpus cavernosal tissue.
Recently, Cartledge et al. reported that glycosylated haemoglobin dramatically impairs endothelial nitric oxide (NO)-mediated relaxation of corpus cavernosum smooth muscle in the rat in vitro. This effect is caused partly by the generation of superoxide anions and the extracellular inactivation of NO; however, the effect in the denervated penis is unclear. Creatine kinase B (spot 1), usually found in nerve tissue, was significantly up-regulated and might be a specific marker of neurogenic ED. Creatine kinase isoenzymes are central to energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa .
It was reported that oxidative stress is important in ED ; in the present study, the expression levels changed in four proteins related to oxidative stress. Factors associated with injury, i.e. NADH dehydrogenase (ubiquinone) 1α subcomplex 10 (spot 10) was up-regulated, whereas factors associated with protection from injury, i.e. aldehyde reductase 1 (spot 11), peroxiredoxin 2 (spot 12) and Cu/Zn superoxide dismutase (SOD, spot 13) were significantly down-regulated. Aldehyde reductase and Cu/Zn SOD are important enzymes for reducing oxidative injury in all tissues. Overexpression of SOD can reduce corporal superoxide anion levels and restore erectile function in diabetic rats . Peroxiredoxins are nuclear-encoded thiol-proteins with molecular masses of 17–24 kDa. They are ubiquitous, catalyse a broad spectrum of peroxides (including H2O2, lipid hydroperoxide, and peroxinitrite), and are related to oxidative stress . The CN is a well-known nonadrenergic noncholinergic nerve that generates NO, which mediates cavernosal smooth muscle cell relaxation and induces penile erection. The role of oxidative stress in the denervated corpus cavernosum warrants further investigation.
The expression levels of certain cytoskeletal proteins changed significantly in the denervated penis. The predominant cell type in the corpus cavernosum, the smooth muscle cell, is central to erectile function. Bilateral CN resection induced significant apoptosis beneath the tunica albuginea, an area largely comprised of smooth muscle cells [5,6]. In the present study some smooth muscle-related proteins, including transgelin (spot 17 and 18), myosin light chain 1 (spot 14), tropomyosin 1α chain (spot 15), and actin cytoplasmic 2 (spot 16), decreased in expression, while others, including myosin regulatory light chain 2 (spot 19; central to regulating smooth muscle) increased .
Myosin light chain is important in regulating muscle contraction, by changing the arrangement of myosin heads, promoting actin–myosin interaction, and regulating myosin ATPase activity. Alteration in myosin light chain expression appears to reflect alteration in heavy chain expression . Transgelin is an actin that cross-links/gels proteins found in fibroblasts and smooth muscle, and is sensitive to transformation and shape changes. Its expression is down-regulated in many cell lines, and this property might be an early and sensitive marker for the onset of transformation . Ablation of transgelin decreases the contractility and actin content of mouse vascular smooth muscle. Thus, transgelin plays a role in contractility, possibly by affecting actin filament organization . Transgelin is very abundant in the penis and was represented by two spots in the present gel image. Both spots of transgelin (differing in Mr/pI ratio because of modification after translation) were down-regulated.
Changes in levels of these contraction proteins in the denervated penis might be due to apoptosis of smooth muscle cells no longer activated by key neurotransmitters from the CN. Thus, these changes affect the relaxation of cavernosal smooth muscle and might lead to ED. It is reasonable to postulate that cavernosal smooth muscle cells rely on neurotrophic mediators.
Another up-regulated protein was gelsolin (spot 20), which can control actin organization by severing filaments, capping filament ends, and nucleating actin assembly. Gelsolins also have specific and apparently discrete roles in several cellular processes, including cell motility, control of apoptosis, and regulation of phagocytosis . Evidence suggests that proteins belonging to the gelsolin super-family might serve as a direct effector of cytoskeletal actin modulation. Cytoskeletal rearrangement occurs in various cellular processes and involves a wide spectrum of proteins. Actin levels were decreased in the present samples, which might be the result of increased gelsolin in the denervated penis.
Annexin-1 (spot 21) directly regulates actin filament assembly. Annexin-1 reduces proliferation by ERK-mediated disruption of the actin cytoskeleton. Overexpression of annexin-1 mediates the disruption of normal cell morphology and inhibits cyclin D1 expression, thereby reducing cell proliferation through proximal modulation of the ERK signal transduction pathway .
Other proteins with significant expression include apolipoprotein A-I (spots 23 and 24) and galectin-7 (spot 22). Changes in apolipoprotein A-I are associated with many diseases, e.g. coronary artery disease and alcoholic liver disease. Our observation of changed galectin-7 (usually found in stratified epithelia and recently found to be expressed in the urethra ) and the report by User et al. of increased smr1 gene expression in the denervated penis, indicate that denervation affects protein expression in the urethra.
In conclusion, the application of proteomics provides an opportunity to assess the molecular nature of smooth muscle, including expression level, post-translation modification, protein–protein interaction, and subcellular location. The present study represents the first application of proteomics to evaluating the denervated penis, and suggests that overall cellular protein changes might represent a sensitive marker for the status of the denervated penis. The expression level of some proteins, especially those involved in cellular energy metabolism, oxidative stress and the cellular cytoskeleton, were significantly modified by CN resection. Although further investigation of these proteins is needed, this preliminary study provides potential clues to the pathophysiological mechanisms underlying neurogenic ED. Further development of this database will provide a valuable resource for the molecular analysis of normal and pathological penile conditions.
We thank Prof Tao-Ming Li from Research Center for Proteome Analysis of Sun Yat-Sen University for the technologic support and mass spectrum.