Structural effects of the slow/b-cardiac myosin heavy chain R453C mutation in cardiac and skeletal muscle

OBJECTIVES: Hypertrophic cardiomyopathy (HCM) represents an important cause of sudden cardiac death particularly in otherwise healthy young individuals. In some families, HCM is caused by distinct mutations of the cardiac beta myosin heavy chain gene (MYH7). DESIGN: We have analyzed the expression of the malignant MYH7Arg453Cys mutation, in cardiac and skeletal muscle, and related it to morphological alterations. RESULTS: Morphological investigation revealed hypertrophic cardiomyocytes but regularly arranged myofibrils. Skeletal muscle showed no sign of structural alterations. CONCLUSIONS: Our results indicate that cardiomyocyte hypertrophy is secondary, due to impaired function, and that the mutation causes no structural alteration in myofibrillar structure in cardiac or skeletal muscle.     

Smooth muscle myosin heavy chains are developmentally regulated in the rabbit bladder

PURPOSE: In smooth muscle (SM), myosin heavy chain (MHC) is expressed predominantly as two isoforms, SM1 and SM2, which are encoded by a single gene and expressed by alternative splicing mechanisms. Although functional differences of these isoforms are unknown, changes in SM1/SM2 ratio have been reported in various pathophysiologic conditions. We analyzed MHC composition of bladder detrusor SM from rabbits of different ages to determine whether SM1 and SM2 isoform expressions are developmentally regulated. MATERIALS AND METHODS: Rabbit bladders on the -11, -4, 1, 7, 14, 21, and 90th days of life were analyzed for SM MHC isoform expression at protein and mRNA levels. Porous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), S1 protection assay, and histological analysis were employed. RESULTS: The predominant MHC isoform in fetal and neonatal bladders was SM1. In the third postnatal week, the SM1/SM2 ratio decreased from 2.3 to 1.0. A stable SM1/SM2 ratio of 0.6 was observed in the adult animal. Although expression of SM1 mRNA was 2.6-fold greater than that of SM2 in the fetus, the relative amount of SM2 mRNA increased rapidly after birth and remained the predominant isoform throughout adult life. Developmental changes in relative amounts of SM1 and SM2 protein in bladder tissues were virtually identical to those of SM1 and SM2 mRNA. SM cell growth and disappearance of primitive mesenchyme from the bladder occurred concomitantly with the MHC isoform shift. CONCLUSIONS: The parallel temporal course of MHC mRNA and protein isoform levels suggests detrusor SM MHC expression may be developmentally regulated at the mRNA level.     

机械牵张对缺血缺氧心肌细胞肌球蛋白重链mRNA表达的影响

目的　探讨机械牵张对缺血缺氧心肌细胞肌球蛋白重链 (MHC)mRNA表达的影响。方法　建立离体培养的SD乳鼠心肌细胞机械牵张模型 ,并施加无糖缺氧刺激因素 ,模拟烧伤后缺血缺氧损害。实验分 10 %牵张组、缺血缺氧培养组、10 %牵张 缺血缺氧培养组 ,每组每时相点 3皿细胞。各组分别在刺激前和刺激后 1、3、6、12h时相点进行观察。采用二步法逆转录聚合酶链反应检测心肌细胞MHCmRNA表达的变化 ,并用凝胶软件进行分析。　结果　与刺激前比较 ,10 %牵张组α、β型MHCmRNA表达均增加 ,但以 β型增加为主 (P <0.0 1)。缺血缺氧培养组α MHCmRNA向 β MHCmRNA转化加速 ,且缺氧 12h后心肌细胞α MHCmRNA下调明显 (P <0.0 5);β MHCmRNA表达先升后降 ,6h表达最强 (P <0.0 5 )。10 %牵张 缺血缺氧培养组α MHCmRNA向 β MHC mRNA转化明显 ,随机械刺激时间的延长转化加剧 ;12h后α、β型MHCmRNA表达均下调 (P <0.0 5)。　结论　机械牵张进一步加剧缺血缺氧对MHCmRNA表达的下调 ,可能是严重烧伤后早期心肌功能降低的原因之一。     

Myocardium expression of connexin 43, SERCA2a, and myosin heavy chain isoforms are preserved in nitrofen-induced congenital diaphragmatic hernia rat model

Background

Previous morphological studies had produced controversial results with regard to heart development in congenital diaphragmatic hernia (CDH), whereas a few publications investigated cardiac function and myocardial maturation. Myocardium maturation is associated with age-dependent increasing of gene expression of gap junction protein connexin 43 (Cx43), adenosine triphosphatase of the sarcoplasmic reticulum (SERCA2a), as well as switching of myosin heavy chains (MHCs) from β to α isoforms. Our aim was to evaluate myocardium maturity in nitrofen-induced CDH rat model.

Methods

Fetuses from dated pregnant Sprague-Dawley rats were assigned to 3 experimental groups: control, nitrofen (exposed to nitrofen, without CDH), and CDH (exposed to nitrofen, with CDH). Myocardial samples collected from left ventricle free wall were processed to (i) quantification of messenger RNA (mRNA) of Cx43, SERCA2a, α and β MHC isoforms, as well as β-actin (housekeeping gene); and (ii) separation of MHC isoforms (α and β isoforms) by sodium dodecyl sulfate polyacrylamide gel electrophoresis silver stained.

Results

We demonstrated that there is no difference in myocardial gene expression of Cx43 (control, 1.0 ± 0.1; nitrofen, 1.1 ± 0.2; CDH, 1.3 ± 0.2) and SERCA2a (control, 1.0 ± 0.1; nitrofen, 0.9 ± 0.1; CDH, 1.0 ± 0.2). Myocardial gene expressions of α and β mRNA of MHC isoforms were slightly decreased both in nitrofen and CDH fetuses when compared with control fetuses, but evaluation of the α-to-β ratios of MHC isoforms at protein level revealed no significant differences between CDH and control (control, 16.9 ± 2.5; CDH, 17.0 ± 2.0).

Conclusions

Myocardial quantification of Cx43 and SERCA2a mRNA, as well as the expression pattern of MHC isoforms at protein levels, was similar in all studied groups. We predict, therefore, that acute heart failure commonly observed in CDH infants might be attributed predominantly to cardiac overload secondary to severe pulmonary hypertension rather than to myocardial immaturity.     

Effect of heart transplantation on skeletal muscle metabolic enzyme reserve and fiber type in end-stage heart failure patients

BACKGROUND: Skeletal muscle myopathy is a hallmark of chronic heart failure (HF). Phenotypic changes involve shift in myosin heavy chain (MHC) fiber type from oxidative, MHC type I, towards more glycolytic MHC IIx fibers, reductions in oxidative enzyme activity, and increase in glycolytic enzyme activity. However, it is unknown if muscle myopathy is reversed following heart transplantation. The purpose of this study was to determine the effect of heart transplantation on skeletal muscle metabolic enzyme reserve and MHC fiber type in end-stage HF patients. METHODS: Thirteen HF subjects were prospectively studied before and two months after heart transplantation and a subgroup (n = 6) at eight months after transplantation. Skeletal muscle biopsy of the vastus lateralis was performed and relative MHC composition was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lactate dehydrogenase (LDH), citrate synthase (CS), and 3-hydroxyacyl-CoA-dehydrogenase (HACoA) enzyme activity assays were performed to assess glycolytic, oxidative, and beta-oxidative metabolic enzyme reserves, respectively. RESULTS: Lactate dehydrogenase activity (130.5 +/- 13.3 vs. 106.1 +/- 13.2 micromol/g wet wt/min, p < 0.05), CS activity (14.0 +/- 1.2 vs. 9 +/- 0.9 micromol/g wet wt/min, p < 0.05), and HACoA activity (4.5 +/- 0.48 vs. 3.6 +/- 0.3 micromol/g wet wt/min, p < 0.05) decreased two months after heart transplantation. At eight months, LDH activity was restored (139.0 +/- 11 micromol/g wet wt/min), but not CS or HACoA activity compared with before transplantation. There was no significant change in muscle %MHC type I (28.7 +/- 3.5% vs. 25.3 +/- 3.0%, p = NS), %MHC type IIa (33.2 +/- 2.0% vs. 34.6 +/- 1.9%, p = NS), or %MHC type IIx (38.1 +/- 2.8% vs. 40.1 +/- 3.7%, p = NS) fiber type two months after heart transplantation. However, %MHC type I (19.3 +/- 6.6%) was decreased and %MHC type IIx (51.0 +/- 6.5%) was increased at eight months after (p < 0.05) compared with before transplantation. CONCLUSIONS: Skeletal muscle glycolytic, oxidative, and beta-oxidative enzymatic reserves are diminished early after heart transplantation, with reduced oxidative capacity persisting late in the first year. The myopathic MHC phenotype present in end-stage HF persists early in the post-operative state and declines further by eight months.     

兔下颌角截骨后咬肌适应性变化

目的通过对兔下颌角弧形截骨后咬肌相关指标测定，研究咬肌在骨架缩短后的变化规律。方法3个月龄雌性新西兰大白兔25只，行左侧下颌角弧形截骨，右侧不手术作为自身对照。2、4、8、12和24周分别处死5只动物，双侧咬肌总量称重。取双侧下颌角中点浅部咬肌，行HE、ATP酶染色。结果左侧咬肌重量减轻与右侧差异显著，肌小节长度2周和4周术侧缩短，8周后恢复正常；部分肌纤维Ⅰ型向Ⅱ型转化；Ⅰ、Ⅱ型肌纤维面积左侧较右侧减小。结论兔下颌角截骨术后肌小节长度、肌纤维转型、咬肌细胞面积发生相应变化，表明下颌骨架缩短后咬肌发生适应性的改建。     

肠道喂养和肠外营养对烫伤大鼠骨骼肌19S调节复合体作用的影响

目的 探讨烧伤患者营养支持的有效途径：方法 分别利用免疫沉淀扣除法，间接ELISA法及荧光分光光度法检测肠道喂养与肠外营养烫伤动物模型的骨骼肌中19S调节复合体的活性和蛋白表达水平及蛋白质降解速率的变化。结果 与肠外营养相比，肠道喂养能明显降低烫伤大鼠骨骼肌中19S调节复合体活性和表达水平，减少骨骼肌蛋白质分解。结论 早期的肠道喂养，可以显著抑制26S蛋白酶复合体系统的活化，从而在整体上降低骨骼肌蛋白质分解代谢，可能有助于烧伤患者的代谢调理。     

Ischemia/reperfusion‐induced necrosis and apoptosis in the cells isolated from rat skeletal muscle

Necrosis was considered to be the solo mechanism for ischemia/reperfusion (I/R)‐induced cell death. Recent evidence from I/R models of the heart, liver, kidney, and brain indicates that apoptosis is a major contributor to I/R‐induced cell death. However, evidence of I/R‐induced apoptosis in skeletal muscle is sparse and divided. The purpose for the present study was to investigate I/R‐induced necrosis and apoptosis in the cells isolated from rat skeletal muscle. A rat gracilis muscle model was used. After surgical preparation, clamps were applied on the vascular pedicle to create 4 h of ischemia and released for 24 h of reperfusion (I/R, n = 10). Clamping was omitted in sham I/R rats (sham I/R, n = 10). The muscle samples were harvested after 24 h of reperfusion for the process of cell isolation. Cells were stained by Propidium Iodide (PI) or Annexin V‐FITC or both. Twenty thousand cells from each muscle sample were scanned and analyzed by flow cytometry. The average percentage of live cells was 45 ± 2% in the I/R group versus 65 ± 3% in the sham I/R group (p < 0.01). The average percentage of necrotic cells was 18 ± 1% in I/R versus 12 ± 1% in sham I/R (p < 0.01). The average percentage of apoptotic cells was 40 ± 3% in I/R versus 27 ± 3% in sham I/R (p < 0.01). Our results clearly demonstrated that I/R not only causes necrosis, but also accelerates apoptosis in the cells isolated from rat skeletal muscle. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:351–356, 2008     

Angiotensin II induces reorganization of the actin cytoskeleton and myosin light-chain phosphorylation in podocytes through rho/ROCK-signaling pathway*

Aims In the present study, we have evaluated the effect of angiotensin II (Ang II) on actin cytoskeleton reorganization and myosin light-chain (MLC) phosphorylation in podocytes to demonstrate whether the Rho/Rho-associated coiled kinase (ROCK) pathway is involved podocyte injury. Methods Eighteen male Sprague–Dawley rats were divided into three groups and treated with Ang II, saline or telmisartan. Morphological changes were studied at 28 days after treatment. Immunohistochemistry and Western blotting were used to determine the renal expression of p-MLC and ROCK2. Cultured podocytes were treated with Ang II (10^?7?M) with or without Rho-kinase inhibitor (Y27632, 10^?6?M) for variable time periods. F-actin was visualized with fluorescein isothiocyanate (FITC)-conjugated phalloidin or tetraethyl rhodamine isothiocyanate (TRITC)-conjugated phalloidin. p-MLC expression was evaluated by immunofluorescence and Western blot. The activation of Rho/ROCK was evaluated by Western blot. Results The expression of p-MLC in glomeruli increased significantly in rats treated with Ang II when compared to the control rats as shown by Western blot (p?Conclusion Ang II-induced podocyte cytoskeleton protein expression changing through the RhoA/ROCK2 p-MLC/F-actin pathway.     

Expression of N-cadherin by skeletal muscle in the degenerationand the degeneration/regeneration processes after nerve injury

We investigated the expression of N-cadherin by skeletal muscle during the degeneration and degeneration/regeneration processes using the rat sciatic nerve and gastrocnemius muscle model. The right sciatic nerve was exposed in the mid-thigh region, and the nerve was transsected with small scissors. After then, nerve was sutured (sutured group), or both edges of the resected nerve were turned and sutured to the muscle of each side (unsutured group). At various periods up to 24 weeks after the operation the middle portion of the gastrocnemius muscle of the treated hindlimbs was removed. Expression of N-cadherin was detected by western blot analysis and immunofluorescent staining with an anti N-cadherin antibody. In the both groups, the degree of expression had already increased by the end of the first postoperative week, but there were no significant differences between the first and second postoperative weeks between the two groups. However, the values recorded at the fourth, sixth, ninth, and twelfth postoperative weeks were significantly higher in the unsutured group than in the sutured group. Immunofluorescent staining was present around the muscular membrane in all specimens including the control. These results indicated that there was a difference in the kinetics of expression of N-cadherin in skeletal muscle between the degeneration and degeneration/regeneration processes of the muscle after injury to the nerve. It was also clear that N-cadherin has a role at the surface of the muscle cell in skeletal muscle, not in the satellite and inflammatory cells, in both groups.     

Clinical application and research progress of extracellular slow wave recording in the gastrointestinal tract

The physiological function of the gastrointestinal (GI) tract is based on the slow wave generated and transmitted by the interstitial cells of Cajal. Extracellular myoelectric recording techniques are often used to record the characteristics and propagation of slow wave and analyze the models of slow wave transmission under physiological and pathological conditions to further explore the mechanism of GI dysfunction. This article reviews the application and research progress of electromyography, bioelectromagnetic technology, and high-resolution mapping in animal and clinical experiments, summarizes the clinical application of GI electrical stimulation therapy, and reviews the electrophysiological research in the biliary system.     

Effects of adrenomedullin and vascular endothelial growth factor on ischemia/reperfusion injury in skeletal muscle in rats

Background

In this study we investigated the effects of adrenomedullin (AM) and vascular endothelial growth factor (VEGF) on skeletal muscle ischemia/reperfusion (I/R) injury in a rat model.

Materials and methods

Thirty-six Wistar rats were randomized into six groups (n = 6). Laparotomy was performed in all groups under general anesthesia. Nothing else was done in Group S (Sham). The Group I/R underwent I/R performed by clamping and declamping of the infrarenal abdominal aorta for 120 min, respectively. Group VEGF and Group AM received intravenous infusion of VEGF (0.8 μg/kg) or AM (12 μg/kg) respectively, without I/R. Group I/R + VEGF and Group I/R + AM received intravenous infusion of VEGF (0.8 μg/kg) or AM (12 μg/kg) immediately after 2 h period of ischemia, respectively. At the end of reperfusion period, skeletal muscle samples of lower extremity were taken from all groups for biochemical and histopathologic examinations.

Results

Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), and hypoxia inducible factor 1 alpha (HIF 1α) were found to be significantly higher in Group I/R than the levels in Group S (P < 0.05). Tissue levels of MDA, SOD, NO, and HIF 1α were significantly lower in Group I/R + AM compared with the levels in Group I/R (P < 0.05). In Group I/R + VEGF, tissue levels of MDA and NO were significantly lower than the levels in Group I/R (P < 0.05). No statistically significant difference was found in the tissue levels of catalase among the groups. Histologic examination revealed a larger central muscular necrosis than the peripheral necrosis, red blood cells in the lumens of capillary vessels, and a stronger atrophy and elliptical or round shape in muscle fibers in Group I/R. Terminal deoxynucleotidyl transferase mediated dUPT nick end labeling (TUNEL)-positive cell count was significantly lower in groups I/R + AM and I/R + VEGF than Group I/R (P < 0.0001, P < 0.0001, respectively).

Conclusions

These results indicate that AM and VEGF have protective effects on I/R injury in skeletal muscle in a rat model.     

The functional and histological effects of clenbuterol on the canine skeletal muscle ventricle

BACKGROUND: We investigated the anabolic effects of the sympatho-mimetic drug clenbuterol upon pumping chambers constructed from latissimus dorsi muscle (LDM). METHODS AND RESULTS: In control and treatment groups (n = 4 dogs each), skeletal muscle ventricles (SMVs) were constructed followed by a 3-week recuperative delay and 6-7 weeks of electrical conditioning at 2 Hz to induce phenotypic expression of fatigue resistant slow muscle fibers. The treatment group received oral administration of clenbuterol (8 microg/kg, 2x/day) during this period. The clenbuterol group increased significantly in body weight as compared with the control group (P < 0.05). In a terminal experiment, the SMVs were assessed with a mock circulation device to determine pumping performance and also were examined with regard to fiber type distribution and area in the SMVs and their contralateral in situ LDMs. Initially the clenbuterol group performed better than the control group, but by the end of a 60-min fatigue test, there were no significant differences. With regard to fiber type distribution and areas, the SMVs of the clenbuterol group exhibited a fast fiber distribution similar to unconditioned muscles (28% +/- 4%), whereas the control group showed complete transformation (100%) to slow fibers. The fast fibers of the clenbuterol group were larger than control (P < 0.05), but the slow fibers were not significantly different. CONCLUSIONS: At the dose given, clenbuterol does induce hypertrophy and preserves the normal percentages of fiber types, possibly by hyperplasia, but it does not affect chronic pumping performance of skeletal muscle ventricles in the canine model.     

Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.

Mutations in the MYH9 gene, which encodes the nonmuscle myosin heavy chain IIA, have been recently reported in three syndromes that share the association of macrothrombocytopenia (MTCP) and leukocyte inclusions: the May-Hegglin anomaly and Sebastian and Fechtner syndromes. Epstein syndrome, which associates inherited sensorineural deafness, glomerular nephritis, and MTCP without leukocyte inclusions, was shown to be genetically linked to the same locus at 22q12.3 to 13. The expression of MYH9 in the fetal and mature human kidney was studied, and the 40 coding exons of the gene were screened by single-strand conformation polymorphism in 12 families presenting with the association of MTCP and nephropathy. MYH9 is expressed in both fetal and mature kidney. During renal development, it is expressed in the late S-shaped body, mostly in its lower part, in the endothelial and the epithelial cell layers. Later, as well as in mature renal tissue, MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells. Four missense heterozygous mutations that are thought to be pathogenic were found in five families, including two families with Epstein syndrome. Three mutations were located in the coiled-coil rod domain of the protein, and one was in the motor domain. Two mutations (E1841K and D1424N) have been reported elsewhere in families with May-Hegglin anomaly. The two others (R1165L and S96L) are new mutations, although one of them affects a codon (R1165), found elsewhere to be mutated in Sebastian syndrome.     

Differential effects of rosiglitazone on skeletal muscle and liver insulin resistance in A-ZIP/F-1 fatless mice

To determine the role of adipocytes and the tissue-specific nature in the insulin sensitizing action of rosiglitazone, we examined the effects of 3 weeks of rosiglitazone treatment on insulin signaling and action during hyperinsulinemic-euglycemic clamps in awake A-ZIP/F-1 (fatless), fat-transplanted fatless, and wild-type littermate mice. We found that 53 and 66% decreases in insulin-stimulated glucose uptake and insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity in skeletal muscle of fatless mice were normalized after rosiglitazone treatment. These effects of rosiglitazone treatment were associated with 50% decreases in triglyceride and fatty acyl-CoA contents in the skeletal muscle of rosiglitazone-treated fatless mice. In contrast, rosiglitazone treatment exacerbated hepatic insulin resistance in the fatless mice and did not affect already reduced IRS-2-associated PI 3-kinase activity in liver. The worsening of insulin action in liver was associated with 30% increases in triglyceride and fatty acyl-CoA contents in the liver of rosiglitazone-treated fatless mice. In conclusion, these data support the hypothesis that rosiglitazone treatment enhanced insulin action in skeletal muscle mostly by its ability to repartition fat away from skeletal muscle.     

慢性传输性便秘模型大鼠血清性激素水平及结肠雌激素受体β的分布与表达研究

目的 研究慢性传输性便秘(STC)大鼠血清性激素水平及结肠雌激素受体β(ERβ)的分布与表达变化.方法 建立STC大鼠模型,以电化学发光法、免疫组化及Western blot技术分别检测STC大鼠与正常大鼠血清性激素及结肠ERβ的分布与表达.结果 电化学发光法检测发现,STC大鼠血清促卵泡生成素(FSH),黄体生成素(LH),雌二醇(E2),孕酮(P)及睾酮(Testo)与正常大鼠比较均无统计学差异(均P＞0.05).免疫组化显示,ERβ主要分布于大鼠结肠的肌间神经丛及黏膜下神经丛,且STC大鼠ERβ的表达明显低于正常大鼠.Western blot显示,STC大鼠结肠ERβ蛋白表达较正常大鼠明显降低,差异有统计学意义(P＜0.0l).结论 STC大鼠结肠ERβ蛋白表达量降低,ERβ表达的改变可能参与STC的发病机制.     

Differential Effects of Bone Structural and Material Properties on Bone Competence in C57BL/6 and C3H/He Inbred Strains of Mice

The femoral neck is a relevant and sensitive site for studying the degree of osteopenia. Engineering principles predict that bone structural parameters, like cross-sectional geometry, are important determinants of bone mechanical parameters. Mechanical parameters are also directly affected by the material properties of the bone tissue. However, the relative importance of structural and material properties is still unknown. The aim of this study was to compare bone competence and structural parameters between a murine strain showing a low bone mass phenotype, C57BL/6 (B6), and another one showing a high bone mass phenotype, C3H/He (C3H), in order to better determine the role of bone structure and geometry in bone failure behavior. Murine femora of 12- and 16-week-old B6 and 12- and 16-week-old C3H inbred strains were mechanically tested under axial loading of the femoral head. In order to assess the structural properties, we performed three-dimensional morphometric analyses in five different compartments of the mouse femur using micro-computed tomography. The mechanical tests revealed that B6 femora became stiffer, stronger, and tougher at 12-16 weeks, while bone brittleness stayed constant. C3H bone stiffness increased, but strength remained constant, work to failure decreased, and bone became more brittle. These age effects indicated that B6 did not reach peak bone properties at 16 weeks of age and C3H did reach maximal skeletal biomechanical properties before 16 weeks of age. Our investigations showed that 83% of the strength of the femoral neck in the B6 strain was explained by cortical thickness at this location; in contrast, in C3H none of the mechanical properties of the femoral neck was explained by bone structural parameters. The relative contributions of bone structural and material properties on bone strength are different in B6 and C3H. We hypothesize that these different contributions are related to differences at the ultrastructural level of bone that affect bone failure.     

The role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury

The aim of this study was to clarify the role of oxygen-derived free radicals and the effect of free radical scavengers on skeletal muscle ischemia/reperfusion injury. Male Wistar rats were divided into a complete ischemia group (C-group) and an incomplete ischemia group (IC-group) and each animal was subjected to 2h of ischemia and 1h of reperfusion. In an attempt to decrease reperfusion injury, the rats were given free radical scavengers either as allopurinol 50 mg/kg for 2 days or as superoxide dismutase 60,000 units/kg plus catalase 500,000 units/kg. Tissue malondialdehyde, a product of lipid peroxidation, was measured as an indicator of free radicals, with higher levels indicating higher concentrations of free radicals. The malondialdehyde level in the gastrocnemius muscle after 1h of reperfusion increased significantly in both groups when compared to the levels before and 2h after ischemia, although there was no significant difference between the two groups. The water content of the gastrocnemius muscle and serum creatinine phosphokinase MM isoenzyme (CPK-MM) in both groups, and GOT in the C-group, increased significantly after 1h of reperfusion when compared the values before and 2h after ischemia. In the C-group, these values were significantly higher than in the IC-group. The administration of free radical scavengers suppressed the increase in malondialdehyde in the gastrocnemius muscle after reperfusion in both groups. The increase in water content and CPK-MM after reperfusion was also suppressed by free radical scavengers in the IC-group, but not in the C-group. These findings suggest that ischemic damage predominates in complete severe ischemia/reperfusion injury, whereas reperfusion injury predominates in incomplete mild ischemia/reperfusion injury.     

Contractile protein expression in bladder smooth muscle is a marker of phenotypic modulation after outlet obstruction in the rabbit model

PURPOSE: We determined changes in contractile protein expression before and after the relief of partial bladder outlet obstruction in the rabbit model and assessed their potential role as predictors of recovery. MATERIALS AND METHODS: We examined the ratio of the smooth muscle myosin heavy chain isoforms SM2-to-SM1, caldesmon isoform expression and bladder function in obstructed and unobstructed adult rabbit bladders. Cystometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were done to determine changes in bladder function and contractile protein expression. RESULTS: Overall we observed significant correlation of bladder weight with the SM2-to-SM1 ratio (p <0.05). Regardless of the duration of obstruction (up to 10 weeks) the ratio appeared to stabilize around a value comparable to that in fetal rabbit smooth muscle cells, suggesting a reversal of SM2 and SM1 expression to a level similar to that at the fetal stage. The pattern of h and l-caldesmon isoform expression showed an increase in l-caldesmon expression in obstructed bladders. Except for decreased leak point pressure in the obstructed group we noted no statistically significant urodynamic changes in bladder capacity or compliance. CONCLUSIONS: There is significant correlation of bladder weight, which is the best known marker of obstruction, with the SM2-to-SM1 ratio. The myosin heavy chain isoform expression ratio appears to be an indicator of phenotypic modulation in bladder smooth muscle before and after the relief of bladder outlet obstruction. Thus, it may be useful as a marker of bladder dysfunction and predictor of functional recovery. Regression to a fetal pattern of protein expression may suggest irreversible damage to smooth muscle cells, possibly limiting recovery.