Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.     

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.     

Preliminary research on myosin light chain kinase in rabbit liver

AIM:To study preliminarily the properties of myosin lightchain kinase(MLCK)in rabbit liver.METHODS:The expression of MLCK was detected byreverse transcdption-polymerase chain reaction(RT-PCR);the MLCK was obtained from rabbit liver,and its activitywas analyzed by γ-~(32) p incorporation technique to detect thephosphorylation of myosin light chain.RESULTS:MLCK was expressed in rabbit liver,and theactivity of the enzyme was similar to rabbit smooth muscleMLCK,and calmodulin-dependent.When the concentrationwas 0.65 mg·L~(-1),the activity was at the highest level.CONCLUSION:MLCK expressed in rabbit liver may catalyzethe phosphorylation of myosin light chain,which may playimportant rolos in the regulation of hepatic cell functions.     

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met¹–Gly⁷⁷³) and the C-terminal long α-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly⁷⁷³–Ser¹¹⁰⁴) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.     

Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibres

Summary We investigated the influence of myosin P-light chain phosphorylation by Ca²⁺-calmodulin dependent myosin light chain kinase (MLCK) on the sensitivity of the tension-pCa relation and maximum unloaded shortening velocity (v _max) of chemically skinned heart fibres of the pig.Submaximum Ca²⁺ stimulation (pCa 5.5) induced 20±5% of the isometric tension achieved at maximum Ca²⁺ activation (pCa 4.3).MLCK-induced myosin P-light chain phosphorylation increased the isometric force development at pCa 5.5 by 40% whereas maximum tension at pCa 4.3 was not affected.Unloaded shortening velocity (v _max) was not altered by myosin P-light chain phosphorylation either at maximum or at submaximum Ca²⁺ concentration, being c. 1.2 muscle length/s at pCa 5.5 and 2.2 muscle length/s at pCa 4.3.The MLCK-induced increase of the myosin P-light chain phosphorylation level was evaluated by determination of³²P-incorporation. Two phosphorylatable myosin P-light chains could be demonstrated.     

Regulation of receptor capping in mouse lymphoma T cells by Ca2+-activated myosin light chain kinase.

Several characteristics of receptor capping in lymphocyte membranes suggest similarities with mechanisms underlying control of contraction in smooth muscle fibers. Both capping and contraction are Ca2+ dependent and require metabolic energy. Contractile proteins such as actin and myosin are associated with the cap, as is calmodulin, which mediates the Ca2+ dependence of smooth muscle contraction. Recent studies have shown that myosin light chain kinase (MLCK), which plays a central role in regulation of smooth muscle contraction, is also present in isolated lymphocyte membrane-cytoskeleton complexes. We have explored this analogy further, using mouse lymphoma T cells whose membranes were rendered permeable to small proteins by using a low-Ca2+ EGTA solution similar to that used to chemically skin smooth muscle cells. Permeabilized lymphocytes were then exposed to solutions containing various combinations of high or low Ca2+, ATP, or other nucleotides (5'-adenylyl imidodiphosphate, adenosine 5'-[gamma-thio]triphosphate, guanosine 5'-[gamma-thio]triphosphate, CTP, ITP, UTP, and GTP), calmodulin, Ca2+-insensitive MLCK (MLCK subunit that has been stripped of the Ca2+ binding site), and the catalytic subunit of cAMP-dependent protein kinase that phosphorylates (and thereby inactivates) MLCK. Capping of concanavalin A-labeled receptors in these various test solutions was scored. In all solutions the capping observed in permeable lymphoma cells correlated well with contraction previously observed in similarly treated skinned smooth muscle fibers, providing strong evidence for the involvement of myosin light chain phosphorylation in the regulation of receptor capping.     

Non-muscle myosin IIA is involved in focal adhesion and actin remodelling controlling glucose-stimulated insulin secretion

Aims/hypothesis

Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS.

Methods

Inhibitors of MLCK, ROCK and NM II were used to study NM II activity, and knockdown of NM IIA and IIB to determine isoform specificity, using sorted primary rat beta cells. Insulin was measured by radioimmunoassay. Protein phosphorylation and subcellular distribution were determined by western blot and confocal immunofluorescence. Dynamic changes were monitored by live cell imaging and total internal reflection fluorescence microscopy using MIN6B1 cells.

Results

NM II and MLCK inhibition decreased GSIS, associated with shortening of peripheral actin stress fibres, and reduced numbers of FAs and insulin granules in close proximity to the basal membrane. By contrast, ROCK inhibition increased GSIS and caused disassembly of glucose-induced central actin stress fibres, resulting in large FAs without any effect on FA number. Only glucose-induced NM IIA reorganisation was blunted by MLCK inhibition. NM IIA knockdown decreased GSIS, levels of FA proteins and glucose-induced extracellular signal-regulated kinase 1/2 phosphorylation.

Conclusions/interpretation

Our data indicate that MLCK–NM IIA may modulate translocation of secretory granules, resulting in enhanced insulin secretion through actin and FA remodelling, and regulation of FA protein levels.     

Increased calcium sensitivity of chemically skinned human atria by myosin light chain kinase

Summary We investigated the influence of myosin P-LC phosphorylation catalysed by calcium/calmodulin-dependent myosin light chain kinase (MLCK) on the tension-pCa relation of chemically skinned human atrial fibres. MLCK-induced increased myosin P-LC phosphorylation sensitized human atrial skinned fibres for calcium by 0.11 pCa-units in patients with valvular heart disease, and by 0.05 to 0.07 pCa-units in patients with coronary heart disease. The MLCK effect could be antagonized by a light chain phosphatase. The protein phosphatase ocadaic acid (OA) had no influence on the tension-pCa relation of skinned human atrial fibres and had no potentiating effect together with MLCK. The MLCK preparation used in this study was from bovine ventricle and revealed a K_M of 1.8×10^–5 M and a V_max of 822 nmol P_i/min/mg using purified bovine ventricular myosin-LCs as substrate.     

Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation

GbaSM-4 cells, smooth muscle cells derived from brain basilar artery, which express both 210-kDa long and 130-kDa short isoforms of myosin light chain kinase (MLCK), were infected with an adenovirus vector carrying a 1.4-kb catalytic portion of MLCK-cDNA in an antisense orientation. Western blot analysis showed that the expression of short MLCK was depressed without affecting long MLCK expression. The contraction of the down-regulated cells was measured by the cell-populated collagen-fiber method. The tension development after stimulation with norepinephrine or was depressed. The additional infection of the down-regulated cells with the adenovirus construct containing the same insert in a sense direction rescued not only the short MLCK expression but also contraction, confirming the physiological role of short MLCK in the contraction. To examine the role of long MLCK in the residual contraction persisting in the short MLCK-deficient cells, long MLCK was further down-regulated by increasing the multiplicity of infection of the antisense construct. The additional down-regulation of long MLCK expression, however, did not alter the residual contraction, ruling out the involvement of long MLCK in the contractile activity. Further, in the cells where short MLCK was down-regulated specifically, the extent of phosphorylation of 20-kDa myosin light chain (MLC20) after the agonist stimulation was not affected. This finding suggests that there are additional factors to MLC20 phosphorylation that contribute to regulate smooth muscle contraction.     

Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles.

Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.     

Myosin light chain kinase inhibitors can block invasion and adhesion of human pancreatic cancer cell lines.

INTRODUCTION: Invasion and metastasis of pancreatic cancer (PC) require cell motility and adhesion, which depend on the activity of cytoskeleton. A cytoskeletal component indispensable for these processes is myosin II, the cytoplasmic analogue of smooth and skeletal muscle myosin. AIMS AND METHODOLOGY: Because the activity of myosin II is accelerated by phosphorylation of myosin II on its regulatory light chain (RLC) by myosin light chain kinase (MLCK), we used two specific MLCK inhibitors, ML-7 and ML-9, for suppression of motility and adhesion of PC cell lines. RESULTS: Both drugs were potent inhibitors, as measured by in vitro motility assay and adhesion assay. When treated with the same concentration of ML-7, the PC cells were rounded up, and the number of stress fibers was reduced markedly. The in vitro migration and adhesion of PC cells were inhibited by ML-7 and ML-9 in a dose-dependent manner, supporting a specific and competitive inhibition of MLCK by these drugs. The inhibition occurred at nontoxic concentrations. CONCLUSIONS: These results highlight the importance of myosin II in the invasion and metastasis of PC cells and suggest the possibility that blocking of myosin II activity by a specific MLCK inhibitor may be a therapeutic strategy for preventing the invasion and metastasis of PC.     

Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 microns in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+ less than 1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+ = 0.5 microM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab).(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of cardiac-specific myosin light chain kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca(2+)/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.     

Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice

The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2^−/−) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2^+/−) mice appeared normal and reproduced well. In SM2^−/− bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2^−/− bladder showed increased contraction to K⁺ depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2^−/− aorta. However, the SM2^−/− bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as α-actin and tropomyosin, were not altered in SM2^−/− bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2^−/− mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology.     

Characterization of antibodies to smooth muscle myosin kinase and their use in localizing myosin kinase in nonmuscle cells.

Antibodies to myosin light chain kinase, purified from turkey gizzard smooth muscle, were developed in rabbits and purified by affinity chromatography on a myosin light chain kinase-Sepharose 4B column. The purified antibodies crossreact with purified smooth muscle myosin light chain kinase but not with a variety of contractile or cytoskeletal proteins. The antibodies inhibit the catalytic activity of smooth muscle myosin light chain kinase and there is an inverse relationship between the kinase activity and the amount of antibody present in an assay. Half-maximal inhibition of myosin kinase activity occurs at an antibody/myosin kinase molar ratio of 10:1. The affinity-purified antibodies to smooth muscle myosin kinase were used to study the location of myosin kinase in a variety of nonmuscle cells. Immunofluorescence studies indicate that myosin light chain kinase is localized on microfilament bundles (stress fibers) in cultured fibroblasts. The stress fiber staining pattern is abolished when the antibodies are incubated with purified smooth muscle myosin light chain kinase prior to staining cells, while the staining pattern is unaffected when the antibodies are incubated with actin, myosin, alpha-actinin, or tropomyosin prior to staining. Moreover, the stress fiber staining pattern is periodic in well-spread gerbil fibroma cells and experiments have demonstrated that myosin light chain kinase appears to have the same periodic distribution as myosin but an antiperiodic distribution relative to alpha-actinin. These data indicate that myosin light chain kinase and its substrate, myosin, are in close proximity and are consistent with the hypothesis that myosin light chain kinase regulates actin-myosin interactions in nonmuscle cells.     

The role of RhoA and Rho-associated kinase in vascular smooth muscle contraction

A variety of contractile agonists trigger activation of the small GTPase RhoA. An important target of activated RhoA in smooth muscle is Rho-associated kinase (ROK), one of the downstream targets that is the myosin binding subunit (MYPT1) of myosin light chain phosphatase (MLCP). Phosphorylation of MYPT1 at T695 by activated ROK results in a decrease in phosphatase activity of MLCP and an increase in myosin light chain (LC20) phosphorylation catalyzed by Ca²⁺/calmodulin-dependent myosin light chain kinase and/or a distinct Ca²⁺-independent kinase. LC20 phosphorylation in turn triggers cross-bridge cycling and force development. ROK also phosphorylates the cytosolic protein CPI-17 (at T38), which thereby becomes a potent inhibitor of MLCP. The RhoA/ROK pathway has been implicated in the tonic phase of force maintenance in response to various agonists, with no evident role in the phasic response, suggesting this pathway as a potential target for antihypertensive therapy. Indeed, ROK inhibitors restore normal blood pressure in several rat hypertensive models.     

Low lung volume alters contractile properties of airway smooth muscle in sheep.

Breathing at volumes lower than functional residual capacity (FRC) can induce changes in nonasthmatic airways consistent with the behaviour of asthmatic airways. This study investigated the chronic effect of breathing at volumes lower than FRC on the contractility of airway smooth muscle and myosin light chain kinase (MLCK) content and activity. Sheep of three age groups (neonate, adolescent and adult) had their FRC reduced by approximately 25%, for 4 weeks using a leather corset. Contractile responses to carbachol were then recorded in isolated tracheal strips and bronchial rings. MLCK content and activity were assessed by immunoblotting. The rate of stress generation increased in the bronchial smooth muscle of both adult and adolescent but not neonatal corseted sheep: adolescent corseted versus control, 65.0 +/- 4.1 versus 103.4 +/- 7.0 s (to reach 50% maximum stress), respectively; and adult corseted versus control, 57.0 +/- 6.4 versus 93.4 +/- 8.2 s, respectively. This was not due to increases in either bronchial or tracheal smooth muscle amount or MLCK content and activity. The present results indicate that chronic breathing at low lung volumes increases the rate of stress generation in airway smooth muscle.     

Alterations of the enteric smooth musculature in diverticular disease

Background

The pathogenesis of diverticular disease (DD) is considered to be multifactorial and involves intestinal motor disturbances and an underlying enteric neuromuscular pathology. While an enteric neuropathy has been well documented, actual studies on concomitant alterations of the enteric musculature are limited. This study is aimed at reassessing the smooth muscle tissue by histological, ultrastructural and molecular-biological approaches.

Methods

Full-thickness sigmoid specimens were obtained from patients with DD (n = 20) and controls (n = 19). Morphometric analysis was performed to evaluate the thickness and connective tissue index of the circular and longitudinal muscle layers as well as the myenteric plexus. Structural alterations were determined by light and transmission electron microscopy. mRNA profiles of components of the contractile smooth muscle apparatus including smooth muscle α-actin, smoothelin, histone deacetylase 8, and smooth muscle myosin heavy chain (SMMHC) were assessed by qPCR. Altered gene expression levels were confirmed at protein level by immunohistochemistry.

Results

Compared to controls, patients with DD showed (1) increased thickness of the circular and longitudinal muscle layers, (2) architectural alterations of smooth muscle cells, (3) increased connective tissue index of the longitudinal muscle layer, (4) focally reduced density of myofilaments at ultrastructural level, (5) specific down-regulation of SMMHC mRNA levels, (6) decreased immunoreactivity of SMMHC, (7) oligo-neuronal hypoganglionosis.

Conclusions

DD is associated with distinct structural and functional alterations of the enteric musculature. The enteric myopathy is characterized by disturbed muscular architecture, connective tissue replacement and loss of specific myofilaments and thus may contribute to the pathogenesis and progression of DD.     

Ischemia/Reperfusion Effects on Bladder Muscle and Mucosa Cell Contractile Regulatory Proteins

Objectives: Ischemia/reperfusion (I/R) has been shown to be the major etiologic factor in animal models in a variety of bladder dysfunctions. Herein we investigate the direct effect of I/R on rabbit bladder contractile regulatory proteins. Methods: Male rabbit bladders were subjected to IR. The contractile responses were recorded and the protein levels of rho‐kinase (ROK), caldesmon (CaD) and myosin light chain kinase (MLCK) were analyzed in both bladder muscle and mucosa. Results: For the mucosa layer, ROK was unchanged after ischemia, but increased significantly after 1 week of reperfusion. MLCK increased after ischemia and then significantly increased after 1 and 2 weeks of reperfusion. In the muscle layer, ROK increased at 2 h of reperfusion, decreased significantly following 1 week of reperfusion, then returned to control level by 2 weeks of reperfusion. MLCK expression significantly decreased as early as ischemia alone and did not recover after reperfusion. For CaD expression in the mucosa layer, both CaD isoforms significantly increased at 1 week of reperfusion, then progressively decreased at 2 weeks of reperfusion. In the muscle layer, both CaD isoforms had different expressions. In the muscle layer, smooth muscle caldesmon (h‐CaD) decreased at ischemia alone and at 2 h of reperfusion, but significantly increased at 2 weeks of reperfusion; whereas non‐muscle caldesmon (l‐CaD) significantly decreased at 2 weeks of reperfusion. Conclusion: As bladder muscle and mucosa have markedly different purposes, it is not surprising that they respond differently to I/R. Bladder mucosa and muscle have different kinds of alternations on contractile regulatory proteins.