Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins

We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC_17b) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins. aorta; mesenteric vessels; calponin; caldesmon     

Blebbistain, a myosin II inhibitor, as a novel strategy to regulate detrusor contractility in a rat model of partial bladder outlet obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40-45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ～50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5' myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3' MHC SM1 and essential light chain isoform MLC(17b) also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.     

Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle

Urinary bladderfilling and emptying requires coordinated control of bladder body andurethral smooth muscles. Bladder dome, midbladder, base, and urethrashowed significant differences in the percentage of 20-kDa myosin lightchain (LC₂₀) phosphorylation (35.45 ± 4.6, 24.7 ± 2.2, 13.6± 2.1, and 12.8 ± 2.7%, respectively) in restingmuscle. Agonist-mediated force was associated with a rise inLC₂₀ phosphorylation, but the extent of phosphorylation atall levels of force was less for urethral than for bladder body smoothmuscle. RT-PCR and quantitative competitive RT-PCR analyses of totalRNA from bladder body and urethral smooth muscles revealed only aslight difference in myosin heavy chain mRNA copy number per total RNA,whereas mRNA copy numbers for NH₂-terminal isoforms SM-B(inserted) and SM-A (noninserted) in these muscles showed a significantdifference (2.28 × 10⁸ vs. 1.68 × 10⁸ for SM-B and 0.12 × 10⁸ vs. 0.42 × 10⁸ for SM-A, respectively), which was also evident atthe protein level. The ratio of COOH-terminal isoforms SM2:SM1 in theurethra was moderately but significantly lower than that in otherregions of the bladder body. A high degree of LC₂₀phosphorylation and SM-B in the bladder body may help to facilitatefast cross-bridge cycling and force generation required for rapidemptying, whereas a lower level of LC₂₀ phosphorylation andthe presence of a higher amount of SM-A in urethral smooth muscle mayhelp to maintain the high basal tone of urethra, required for urinary continence.

     

The expression and functional activities of smooth muscle myosin and non‐muscle myosin isoforms in rat prostate

Benign prostatic hyperplasia (BPH) is mainly caused by increased prostatic smooth muscle (SM) tone and volume. SM myosin (SMM) and non‐muscle myosin (NMM) play important roles in mediating SM tone and cell proliferation, but these molecules have been less studied in the prostate. Rat prostate and cultured primary human prostate SM and epithelial cells were utilized. In vitro organ bath studies were performed to explore contractility of rat prostate. SMM isoforms, including SM myosin heavy chain (MHC) isoforms (SM1/2 and SM‐A/B) and myosin light chain 17 isoforms (LC_17a/b), and isoform ratios were determined via competitive RT‐PCR. SM MHC and NM MHC isoforms (NMMHC‐A, NMMHC‐B and NMMHC‐C) were further analysed via Western blotting and immunofluorescence microscopy. Prostatic SM generated significant force induced by phenylephrine with an intermediate tonicity between phasic bladder and tonic aorta type contractility. Correlating with this kind of intermediate tonicity, rat prostate mainly expressed LC_17a and SM1 but with relatively equal expression of SM‐A/SM‐B at the mRNA level. Meanwhile, isoforms of NMMHC‐A, B, C were also abundantly present in rat prostate with SMM present only in the stroma, while NMMHC‐A, B, C were present both in the stroma and endothelial. Additionally, the SMM selective inhibitor blebbistatin could potently relax phenylephrine pre‐contracted prostate SM. In conclusion, our novel data demonstrated the expression and functional activities of SMM and NMM isoforms in the rat prostate. It is suggested that the isoforms of SMM and NMM could play important roles in BPH development and bladder outlet obstruction.     

Shortening velocity and myosin heavy- and light-chain isoform mRNA in rabbit arterial smooth muscle cells

In smooth muscle cells (SMCs)isolated from rabbit carotid, femoral, and saphenous arteries, relativemyosin isoform mRNA levels were measured in RT-PCR to test forcorrelations between myosin isoform expression and unloaded shorteningvelocity. Unloaded shortening velocity and percent smooth muscle myosinheavy chain 2 (SM2) and myosin light chain 17b(MLC_17b) mRNA levels were not significantly different insingle SMCs isolated from the luminal and adluminal regions of thecarotid media. Saphenous artery SMCs shortened significantly faster(P < 0.05) than femoral SMCs and had more SM2 mRNA(P < 0.05) than carotid SMCs and lessMLC_17b mRNA (P < 0.001) and higher tissuelevels of SMB mRNA (P < 0.05) than carotid and femoralSMCs. No correlations were found between percent SM2 and percentMLC_17b mRNA levels and unloaded shortening velocity in SMCsfrom these arteries. We have previously shown that myosin heavy chain(MHC) SM1/SM2 and SMA/SMB and MLC_17a/MLC_17b isoform mRNA levels correlate with protein expression for these isoforms in rabbit smooth muscle tissues. Thus we interpret these results to suggest that 1) SMC myosin isoform expression andunloaded shortening velocity do not vary with distance from the lumenof the carotid artery but do vary in arteries located longitudinally within the arterial tree, 2) MHC SM1/SM2 and/orMLC_17a/MLC_17b isoform expression does notcorrelate with unloaded shortening velocity, and 3)intracellular expression of the MHC SM1/SM2 and MLC_17a/MLC_17b isoforms is not coregulated.

     

Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Effect of aging on the response of biochemical markers in the rabbit subjected to short-term partial bladder obstruction

Purpose Partial bladder outlet obstruction (PBOO) results in marked biochemical alterations in the bladder. In this study, we focused on comparison of thapsigargin sensitive sarco/endoplasmic reticulum Ca²⁺ ATPase activity (SERCA) and Citrate Synthase after short term PBOO in young versus old rabbits. Materials and methods A total of 20 young and 20 mature male rabbits were divided into 4 sub-groups of 5 rabbits each (4 obstructed and 1 sham-control rabbit). The rabbits in the groups were evaluated after 1, 3, 7, and 14 days of obstruction, respectively. The activities of SERCA and citrate synthase were examined as markers for sarcoplasmic reticular calcium storage and release and mitochondrial function, respectively. Results The SERCA activity of bladder body smooth muscle in the young animals increased at 7 and 14 days. For the old rabbits, the SERCA activity decreased significantly by 1 day and remained this level throughout the course of obstruction, and was significantly lower than young at all time periods. The citrate synthase activity in the young animals decreased over the 1–7 days, and then returned toward control level by 14 days following obstruction. In the old animals, citrate synthase activity of bladder body smooth muscle progressively decreased over the course of the study, and was significantly lower in the old than the young animals after 14 days obstructed. Conclusion The urinary bladders of the young rabbits have a considerable greater ability to adapt to PBOO than do those of the old rabbits. The deterioration of mitochondrial and SR function may be important mechanisms underlying geriatric voiding dysfunction.     

Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction

The sarco(endo)plasmic reticulum Ca(2+)-ATPase2 (SERCA2) is downregulated in cardiac hypertrophy with decompensation. We sought to determine whether mice heterozygous for the SERCA2 allele would develop greater bladder hypertrophy and decompensation than their wild-type littermates following partial bladder outlet obstruction (pBOO). We found that following 4 wk of surgically created pBOO, SERCA2 heterozygous murine bladders showed significantly less hypertrophy, improved in vitro cystometry performance, diminished expression of the slow myosin isoform A analyzed by RT-PCR, a significant drop in nuclear translocation of nuclear factor of activated T cells by EMSA, and decreased cell proliferation within the smooth muscle layer following 5-bromo-2'-deoxyuridine labeling compared with their wild-type littermates. Thus, in contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy in a murine model of pBOO. Compensatory mechanisms in heterozygous mice seem to be related to the calcineurin pathway. Further studies are underway to better define the molecular basis of this observation, which has potential clinical applications.     

Pathophysiology of benign prostatic hyperplasia in the aging male population

Nearly all men will develop histological benign prostatic hyperplasia by the age of 80, but the degree of prostatic enlargement resulting from the hyperplasia is highly variable. Historically, it has often been assumed that the pathophysiology of lower urinary tract symptoms (LUTS) in men is the result of bladder outlet obstruction associated with prostatic enlargement. The observation that prostatic enlargement, bladder outlet obstruction, and LUTS are all age-dependent has been interpreted to indicate that these phenomena were causally related, but there is insufficient evidence for this. Undoubtedly, some men' prostatic enlargement causes obstruction and symptoms. Based upon the available data, however, this subset appears to be extremely small. Because of the many urological and nonurological conditions that cause LUTS and age-dependent changes in bladder and neurological function, it is unlikely that there exists a single dominant etiology for the aging male population. If this is the case, then the optimal management of LUTS will require different and possibly combination therapies.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

Estrogen modulates the expression of myosin heavy chain in detrusor smooth muscle

The effect of low serum estrogen levels on urinary bladder function remains poorly understood. Using a rabbit model, we analyzed the effects of estrogen on the expression of the isoforms of myosin, the molecular motor for muscle contraction, in detrusor smooth muscle. Expression of myosin heavy chain (MHC) isoforms, which differ in the COOH-terminal (SM1 and SM2) and the NH(2)-terminal (SM-A and SM-B) regions as a result of alternative splicing of the mRNA at either the 3'- or 5'-ends, was analyzed in age-matched female rabbits that were sham operated, ovariectomized (Ovx), and given estrogen after ovariectomy (4 rabbits/group). Ovx rabbits showed a significant decrease in the overall MHC content per gram of wet detrusor smooth muscle compared with controls (P < 0.04), which was reversed by estrogen replacement (P < 0.02). MHC content, as a proportion of total milligram of protein in the bladder tissue extracted, was also increased in estrogen-treated Ovx rabbits. Quantitative competitive RT-PCR revealed 1.72-, 2.63-, and 5.82 x 10(6) copies of MHC mRNA/100 ng total mRNA in Ovx, control, and estrogen-treated rabbits, respectively (P < 0.01). RT-PCR analysis using oligonucleotides specific for the region containing the SM1/SM2 MHC alternative splice sites indicated a lower SM2-to-SM1 ratio in estrogen-treated compared with control and Ovx rabbits (P < 0.05). Similarly, SDS-PAGE analysis of extracted myosin from estrogen-treated rabbits revealed a significantly lower SM2-to-SM1 isoform ratio compared with control and Ovx rabbits (P < 0.05). Expression of the SM-A and SM-B isoforms was not affected. These results indicate that myosin content is increased upon estrogen replacement in Ovx rabbits and that the abundance of SM1 relative to SM2 is greater in estrogen-treated rabbits compared with normal and Ovx rabbits. These data suggest that estrogen affects alternative splicing at the 3'-end of the MHC pre-mRNA to increase the proportion of SM1 vs. SM2.     

Effects of L-arginine and L-NAME on chronic partial bladder outlet obstruction in rabbit

Nitric oxide (NO) is synthesized from L-arginine by nitric oxide synthase (NOS). NOS can be inhibited by NG-nitro-L-arginine methyl ester (L-NAME) and stimulated by supplementing the diet with L-arginine. The aim of this study was to investigate the influence of NOS activity on the response of rabbits to chronic partial bladder outlet obstruction (PBOO). Surgical PBOOs (2 and 8 wk) were performed on male New Zealand White rabbits. Before obstruction, one-third of the animals were premedicated for 7 days with L-NAME and another third with L-arginine. The results are summarized as follows. First, bladder weight after 8-wk PBOO was significantly lower in animals treated with L-arginine compared with both untreated and rabbits treated with L-NAME. Second, contractile function decreased progressively with PBOO duration. However, after 8 wk of PBOO, the L-arginine group had significantly greater contractile function compared with the no-treatment group, and the L-NAME group had significantly lower contractile function compared with the no-treatment group. Third, at 8 wk following PBOO, the level of protein oxidation and nitration was lowest for the L-arginine group and highest in the L-NAME group. These studies clearly demonstrated that increasing blood flow by stimulating NOS significantly protected the bladder from PBOO dysfunctions, whereas inhibiting blood flow by L-NAME enhanced the dysfunctions mediated by PBOO.     

Expression of smooth muscle myosin light chain 17 and unloaded shortening in single smooth muscle cells

These experiments were performed totest the hypotheses that myosin light chain 17 (MLC₁₇) aand b isoform expression varies between individual vascular smoothmuscle (SM) cells and that their expression correlates with cellunloaded shortening velocity. Single SM cells isolated from rabbitaorta and carotid arteries were used to measure unloaded shorteningvelocity and subsequently were analyzed via RT-PCR forMLC₁₇ a and b mRNA ratio. The MLC_17b/a mRNA andprotein ratios from adjacent tissue sections correlate very well(R² = 0.68), allowing use of the mRNA ratio topredict the protein ratio. The rabbit MLC₁₇ isoform proteinsequence was found to be similar to, but unique from, the swine, mouse,and chicken sequences. Isolated single SM cells from the aorta andcarotid have resting lengths of 70-280 µm and shorten to33-88 µm after contraction. Isolated cell maximum unloadedshortening velocity is highly variable (0.5-7.5 µm/s) butbecomes more uniform when normalized to initial cell length(0.01-0.05 cell lengths/s). Carotid cells activated in thepresence of okadaic acid (1 µm) have mean maximal unloaded shorteningvelocities not significantly different from carotid cells activatedwithout okadaic acid (0.016 vs. 0.019 cell lengths/s). Resting celllength before activation is significantly correlated with final celllength after unloaded shortening. Neither initial cell length, finalcell length, total cell length change, nor maximum unloaded shorteningvelocity (absolute or normalized) was significantly correlated withsingle-cell MLC_17b/a mRNA ratio. These studies wereperformed in isolated single SM cells where unloaded shorteningvelocity and MLC_17b/a mRNA ratios were measured in the samecell. In this preparation, the three-dimensional organization andmilieu of the cell is kept intact, but without the intercellularheterogeneity concerns of multicellular preparations. These resultssuggest the MLC_17b/a ratio is variable between individual SM cells from the same tissue, but it is not a determinant of unloadedshortening velocity in single SM cells.

     

Mitochondrial involvement in bladder function and dysfunction

Benign bladder pathology resulting from prostatic hypertrophy or other causes is a significant problem associated with ageing in humans. This condition is characterized by increased bladder mass, decreased urinary flow rate, decreased compliance, and these and other changes in bladder function often subject patients to increased risk of urinary tract infection. While the physiologic attributes of benign bladder pathology have been extensively described in humans and in various animal model systems, the biochemical and molecular genetic bases for that pathology have only recently been investigated in detail. Studies demonstrate that mitochondrial energy production and utilization are severely impaired in bladder smooth muscle during benign bladder disease, and to a large extent this realization has provided a rational basis for understanding the characteristic alterations in urinary flow and compliance in bladder tissue. Recent investigations targeting the detailed molecular basis for impaired mitochondrial function in the disease have shown that performance of the organellar genetic system, and to a large extent that of relevant portions of the nuclear genetic system as well, is severely aberrant in bladder tissue. In this article, we discuss the physiologic aspects of benign bladder disease, summarize biochemical evidence for the altered mitochondrial energy metabolism that appears to underlie bladder pathology, review the structure and function of the mitochondrial genetic system, and discuss molecular genetic studies of that system which have begun to provide a mechanistic explanation for the biochemical and physiological abnormalities that characterize the disease. We also discuss areas for further research which will be critically important in increasing our understanding of the detailed causes of benign bladder pathology.     

Estrogens and bladder outlet obstruction

Increasing evidence indicates a direct interrelationship between benign prostatic hyperplasia and chronic non-bacterial prostatic inflammation in the development of human voiding dysfunction in aging male, which gradually transforms to bladder outlet obstruction (BOO). Increased prevalence of BOO along with the aging process further suggests that estrogen or more precisely decreased androgen to estrogen ratio in serum is involved in the pathogenesis of BOO. In this review, we will analyze the hormonal causes, clinical relevance, and biologically relevant estrogen-modulated animal models potential for BOO study. In light of the data presented in this review, it becomes apparent that direct inhibition of estrogen action may provide important pharmaceutical treatment of the BOO.