Age-related changes in gap junctional protein of the rat heart

OBJECTIVE:

Connexin 43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in physiological processes such as tissue homeostasis, growth regulation and development. The aim of the present study was to investigate the change of Cx43 expression in aged myocardium.

METHODS AND RESULTS:

Sixteen male Sprague-Dawley rats (adult: 10 weeks old, n=8; aged: two years old, n=8) were used in the present study. In an isolated rat heart Langendorff model, hearts were perfused for 10 min with a modified Krebs-Henseleit bicarbonate buffer. Contractile functions were measured and all hearts were stained with anti-Cx43 antibody for fluorescence microscopic examinations. There were no significant differences observed in heart rate (234±8.2 beats/min versus 231±15.6 beats/min), left ventricular developed pressure (112.5±6.3 mmHg versus 107.2±2.5 mmHg), first derivative of the left ventricular pressure (1450.4±165.1 mmHg/s versus 1384.6±95.4 mmHg/s) and coronary flow (17.4±0.7 mL/min versus 21.3±1.8 mL/min) between adult and aged rats, respectively. However, significant differences were observed in left ventricular weight (adult versus aged; 0.639±0.108 g versus 1.124±0.257 g, P=0.04) and in fluorescence examinations where there was reduced distribution of Cx43 in aged myocardium compared with adult myocardium.

CONCLUSIONS:

These results demonstrated that the role of Cx43 may be more important than previously reported, and that this protein is partially responsible for the maintenance of cellular structure in myocardial development.     

Differential regulation of apoptosis in slow and fast twitch muscles of aged female F344BN rats

Age-related muscle atrophy is characterized by decreases in muscle mass and is thought be mediated, at least in part, by increases in myocyte apoptosis. Recent data has demonstrated that the degree of muscle loss with aging may differ between males and females while other work has suggested that apoptosis as indicated by DNA fragmentation may be regulated differently in fast- and slow-twitch muscles. Herein, we investigate how aging affects the regulation of muscle apoptosis in the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles of young (6-month), aged (26-month), and very aged (30-month) female Fischer 344/NNiaHSD × Brown Norway/BiNia (F344BN) rats. Tissue sections were stained with hydroethidium for ROS and protein extract was subjected to immunoblotting for assessing apoptotic markers. Our data suggest that decreases in muscle mass were associated with increased DNA fragmentation (TUNEL positive) and increases in reactive oxygen species (ROS) as determined by hydroethidium staining in both the EDL and soleus. Similar to our previous work using aged male animals, we observed that the time course and magnitude of changes in Bax, Bcl-2, caspase-3, caspase-9, and cleavage of α-fodrin protein were regulated differently between muscles. These data suggest that aging in the female F344BN rat is associated with decreases in muscle mass, elevations in ROS level, increased muscle cell DNA fragmentation, and alterations in cell membrane integrity and that apoptotic mechanisms may differ between fiber types.     

Aging change of mandibular condyle in female F344/N rat

To ascertain whether laboratory rats represent an adequate animal model for aging oral cavity research, this study focused on the morphology of the mandibular condyle. Aging changes of cartilaginous conformation and shape of the mandibular condyles were analyzed in female F344/N rats. In the condylar cartilage, articular, proliferative cell and hypertrophic cell layers were observed in 1-month-old (M) rat, whereas flattened cell layer was notable at 2 M. A mature cell layer was observed in the condylar cartilage of rats at 7.7 M and over. Deranged cartilaginous layers and thinning articular layer were observed in 30.9 M rat. The sagittal length of the condyle decreased, whereas the frontal one increased with aging and/or age. There were three phases in the transition patterns of the size of the condyle, which seemed to correspond to the respective growing, aging and senescence phases in the rat. The results suggested that degenerative change of condylar cartilage in rat was similar to that in human, whereas change of the shape of the condyle was different between rat and human, caused by a different pattern of mastication.     

Age-associated changes in function, structure and mitochondrial genetic and enzymatic abnormalities in the Fischer 344 x Brown Norway F(1) hybrid rat heart.

We hypothesized that cardiac aging in the rat involves mitochondrial genetic damage and mitochondrial enzymatic dysfunction of individual cardiomyocytes as has been demonstrated previously only in primate myocardium. Myocardium from Fischer 344 x Brown Norway F(1)hybrid rats of ages 5, 18 and 36-38 months was examined for mitochondrial genetic and enzymatic abnormalities. In-vivo hemodynamic measurements revealed age-related changes of left ventricular function while histological evaluation demonstrated an increase in percent area fibrosis from 7%+/-5 in the 5-month-old hearts to 38%+/-2 in the subendocardium of the left ventricle of 38-month-old rats. Mitochondrial genomes lacking 8000 to 9000 bp of primary sequence were detected in tissue homogenates from right and left ventricular myocardium and the abundance of these deleted genomes increased with age. In-situ histochemical staining of serial cryomicrotome sections of myocardial tissue revealed individual cardiomyocytes displaying abnormal, primarily absent, activities of cytochrome c oxidase and succinate dehydrogenase. The area density of histochemically-abnormal cardiomyocytes increased from 0.05 per mm(2)to 0.3 per mm(2)between 5 and 36-38 months of age in the left ventricle, and they were localized primarily to the left ventricular subendocardium. The presence of age-related mitochondrial genetic and enzymatic abnormalities in the Fischer 344 x Brown Norway F(1)hybrid rat heart suggests the role of mitochondrial dysfunction, secondary to mtDNA mutations, in age-related cardiomyocyte loss and subsequent cardiac aging.     

Aging changes in the periodontal bone of F344/N rat

The aim of this research was to determine whether a rat was an adequate laboratory animal model for periodontal research on elderly humans. Thirty-two F344/NSlc female rats ranged between 30 and 1000 days of age were used. The alveolar bone loss around the molars was assessed by a morphometric method. A significant correlation was found between age and the amount of alveolar bone loss. For further analysis, the rats were grouped into four by age; 30–60 days, 220–430 days, 640–850 days, and more than 850 days. The means of alveolar bone loss were compared between age groups. It was found that the resorption of the alveolar bone around the molars of the rats continued until they were 1000-days-old, and this trend was stronger in the mandible than the maxilla. It was suggested that rats could be used as adequate laboratory animals for periodontal research.     

心脏结构和功能随增龄变化的超声心动图观察

目的 观测心脏结构和功能的增龄性改变及部分影响因素。方法 对306例高校健康教师行超声心动图检查，年龄30－85岁，男165人，女141人。结果 在男女两性，二尖瓣频谱E峰与A峰比值（E／A）、三尖瓣频谱E峰与A峰比值（E／Ar)、主动脉运动幅度（Aao)、室间隔与主动脉根部的夹角（θ）随增龄显著下降（P＜0．001），等容舒张时间（IVRT）随增龄显著延长（P＜0．001），左房前后径（LAD）随增龄显著增大（男P＜0．001，女P＜0．01），左室后壁舒张末期厚度（LVPWTd)及升主动脉径（AOD）在女性随增龄也显著增加（P＜0．05），在男性随龄变化不显著。多因素分析显示，年龄、体重指数（BMI）是E／A、E／Ar、IVRT的独立影响因素；BMI对AOD、LVPWTd及LAD的影响大于年龄，BMI大者E／A减低，IVRT、AOD、LVPWTd及LAD趋于增加。结论 伴随增龄，左右心室舒张功能减低，室间隔形态呈乙状改变，Aao减低，左房、主动脉径增大，左室后壁趋于增厚。女性随增龄室壁厚度的增加较男性显著，肥胖可加速伴随增龄心脏结构及功能的改变。     

Diabetes-induced alterations of reproductive and adrenal function in the female rat

Diabetes interferes with reproductive function in laboratory animals. Previous studies in female diabetic rats have not resolved if the reproductive abnormalities observed are at the hypothalamic, pituitary and/or ovarian level. The interaction of the gonadal and adrenal axes has not been studied in the diabetic female rat. The purpose of this study is twofold: first, to determine the level of dysfunction in the hypothalamic-pituitary axis caused by diabetes in the adult female rat controlling for stage of the estrous cycle, and, second, to evaluate basal corticosterone secretion in female diabetic rats. Sixty cycling 40-day-old female rats were randomly assigned to 3 groups; control (n = 32), diabetic (n = 14), and diabetic insulin-replaced animals (n = 14). The level of hyperglycemia in each group was documented by glycosylated hemoglobin levels and biweekly blood glucoses. Three weeks after induction of diabetes, pituitary luteinizing hormone (LH) responsiveness following an i.v. injection of gonadotropin-releasing hormone (GnRH) was assessed in representative diestrous rats from each group. All animals were sacrificed in either diestrus or proestrus for determination of GnRH concentration in the hypothalamus, LH and follicle-stimulating hormone (FSH) content in pituitary and LH, FSH, estradiol and corticosterone in serum. Uterine weight to body weight ratios (a bioassay for estrogen) were also calculated. Hypothalamic GnRH concentration was significantly lower in diabetic versus control diestrous rats. Basal pituitary and serum gonadotropin levels were not different between any groups. GnRH-stimulated serum LH levels were higher in diabetic vs. control and diabetic insulin-treated animals. LH surges occurred in the control and diabetic insulin-replaced but not the diabetic group.(ABSTRACT TRUNCATED AT 250 WORDS)     

肥胖对中青年人群心脏结构和功能的影响

目的　研究肥胖对我国中青年人群心脏结构和功能的影响。方法　选择 6 7例肥胖者和 2 3名健康者。肥胖者根据体重指数 (BMI)分为三组。采用美国HDI30 0 0彩色多普勒超声心动图仪测量 ,比较各组间心脏结构 (收缩末期左室内径 (LVESD)、舒张末期左室内径 (LVEDD)、室间隔 (IVS)及左室后壁 (LVPW )厚度 ,左室重量 (LVM )和左室重量指数 (LVMI) )和功能 (左室每搏量指数 (SVI) ,射血分数 (EF) ,舒张早期血流充盈峰值流速E、舒张晚期充盈峰值流速A及E/A ,并测量等容舒张时间 (IVRT) )参数。结果　肥胖组同健康组比较 ,LVEDD、LVESD增宽 ,IVS、LVPW增厚 ,LVM、LVMI增加 ,舒张早期充盈峰值流速E降低 ,舒张晚期充盈峰值流速A升高 ,E/A比值降低 ,IVRT延长 ,差异有显著性 (P <0 0 5 )。并随肥胖程度的加重有加重趋势。肥胖组收缩功能参数与健康组比较变化无显著性 (P >0 0 5 )。肥胖组体重指数与LVEDD显著正相关 (r =0 37,P <0 0 5 ) ,与LVM显著正相关 (r =0 5 3,P <0 0 5 ) ,与E/A峰速度比值显著负相关 (r =- 0 2 7,P <0 0 5 )。结论　肥胖可引起中青年人群心脏肥大 ,使中青年人群心脏舒张功能下降 ,并随肥胖程度而加重。     

缝隙连接蛋白43在去卵巢大鼠骨细胞中的表达及其与骨质疏松的关系

目的　探讨去卵巢致骨质疏松大鼠骨细胞中缝隙连接蛋白 4 3(Cx4 3)的表达及意义。　方法　采用 10月龄未孕产Wistar雌性大鼠 30只 ,随机分为去卵巢组、假手术组和尼尔雌醇治疗组。于术后 8周末测量 3组大鼠全身及腰椎骨密度 (BMD) ,采用放免法测定血清雌二醇水平 ,采用SABC免疫组化法观察Cx4 3在成骨细胞和破骨细胞中的表达情况 ,采用 3’ OH末端DNA原位标记技术观察凋亡细胞变化。　结果　术后 8周末去卵巢组全身及腰椎BMD和血清雌二醇水平明显低于假手术组和尼尔雌醇治疗组 (P <0 0 1)。去卵巢组成骨细胞内Cx4 3阳性表达率 (13 80 %±1 14 % )低于假手术组 (6 3 6 0 %± 2 4 6 % )和尼尔雌醇治疗组 (6 3 6 0 %± 2 12 % ) (P <0 0 1) ,而破骨细胞内去卵巢组Cx4 3阳性表达率 (6 6 10 %± 1 37% )高于假手术组 (42 2 0 %± 1 93% )和尼尔雌醇治疗组 (41 80 %± 1 81% ) (P <0 0 1)。去卵巢组成骨细胞的凋亡变化 (49 30 %± 3 86 % )高于假手术组 (2 4 2 0 %± 2 78% )和尼尔雌醇治疗组 (2 5 5 0 %± 3 0 3% ) (P <0 0 1) ,破骨细胞凋亡变化(5 80 %± 1 14 % )低于假手术组 (19 2 0 %± 1 75 % )和尼尔雌醇治疗组 (19 70 %± 1 6 9% ) (P <0 0 1)。而上述观察指标中假手术组和尼尔雌醇治疗     

实验性糖尿病心肌病变心肌连接蛋白43的意义

免疫组化染色显示,正常大鼠的心肌间隙连接蛋白43(Cx43)大量表达(平均灰度值230±5),分布在相邻细胞的连接处和闰盘部位。STZ诱导的糖尿病大鼠的Cx43表达明显减少(平均灰度值193±4)(P<0·01),且分布紊乱。这可能相关于糖尿病心肌病变的发病。     

Glial glucocorticoid receptors in aged Fisher 344 (F344) and F344/Brown Norway rats

Glucocorticoid receptors (GR) regulate glial function, and changes in astrocyte gene expression are implicated in age-related pathology. We evaluated changes in astroglial GR expression in two strains of rats – Fisher 344 (F344; 4, 12 and 24 months) and F344/Brown Norway strain (F344/BN; 4, 12 and 30 months). In both strains basal levels of corticosterone were higher in the oldest groups of rats. Age-related increases in GR (+) astrocytes but not the percent of astrocytes expressing GR were observed in the hippocampus CA1 region in F344 rats. Age-related decreases in CA1 GR (+) astrocytes and the percentage of GR (+) astrocytes were observed in the F344/BN strain only. Similar strain-specific changes were observed in the dentate gyrus. In the hypothalamic paraventricular nucleus: (1) F344 rats exhibited significant decreases in the overall number of glial profiles with age, (2) F344/BN rats exhibited decreases in the numbers of GR (+) astrocytes with aging and (3) the proportion of GR (+) astrocytes decreased in older F344/BN, but not F344 rats. Overall, the data demonstrate age- and strain-related alterations in GR astrocytic expression that may explain unique phenotypic differences in brain function observed in both strains.     

不同β受体阻滞剂对大鼠心肌间隙连接结构作用的对比研究

目的比较三代β受体阻滞剂的代表药物卡维地洛、美托洛尔及普萘洛尔对大鼠心肌缺血再灌注损伤心肌间隙连接（GJ）结构的不同作用。方法将大鼠随机分为假手术组、缺血再灌注组、卡维地洛组、美托洛尔组及普萘洛尔组。除假手术组只穿线不结扎外,其余各组均结扎左冠状动脉前降支30min,然后松开结扎线复灌4h,建立心肌缺血再灌注损伤模型。于再灌4h末用免疫荧光和激光扫描共聚焦显微镜技术观察心肌间隙连接蛋白43（CX43）的分布及组成变化,用激光扫描共聚焦显微镜对CX43进行定量。结果与假手术组相比,缺血再灌注组CX43-GJ结构明显异常。与缺血再灌注组比较,卡维地洛组、美托洛尔组和普萘洛尔组CX43-GJ损伤减轻。各药物治疗组间比较,卡维地洛组CX43-GJ结构损伤最轻。结论各种β受体阻滞剂均具有保护心肌GJ结构的作用,以卡维地洛的作用最明显。     

利用超声心动图评定大鼠心脏功能的可行性研究

目的 :通过与血流动力学检测比较 ,确定 M-型超声心动图是否可用来评价正常和心肌梗死大鼠左心室功能和结构变化。方法　利用冠状动脉左前降支结扎术制备大鼠大面积心肌梗死模型后 ,术后 3 d和 3 0 d进行 M-型超声心动图测定、血流动力学检测和取材称左心室重量 ;同时也对心脏功能正常大鼠进行上述研究。结果　用 M-型超声心动图测定心脏功能正常大鼠 ( NOR组 )、心肌梗死急性心力衰竭大鼠 ( AHF组 )和慢性充血性心力衰竭大鼠( CHF组 )的 EF和 FS值与用血流动力学方法测得的 LVdp/ dtmax值呈良好的正相关 ( r=0 .811-0 .972 ,均 P<0 .0 1) ;心肌梗死后 3 d和 1月 EF值与 FS值变化趋势与 L Vdp/ dtmax值变化趋势一样 ,均明显低于 NOR组 (均 P<0 .0 1) ,而且 CHF组各值也明显低于 AHF组 (均 P<0 .0 1)。同时还观察到应用超声心动图测得的三种心功能状态大鼠左心室重量与精密天平检测结果基本一致 (均 P>0 .5 )。结论　无创性经胸壁二维引导 M-型超声心动图可用来动态评价心肌梗死引起的大鼠心脏功能和结构改变     

Regional variation in cardiac myosin isoforms of female F344 rats during aging.

Myosin heavy chain (MHC) is a major contractile protein of heart muscle consisting of two isoforms in the rat, alpha-MHC that predominates in the hearts of young rats, and beta-MHC that progressively replaces it as the rats age. It was hypothesized that the magnitude of the age-associated decrease in the proportion of cardiac alpha-MHC would be similar in regions of the heart that differed in their initial MHC isoform pattern. MHCs from hearts of female Fischer 344 rats 3, 9, 15, 18, 24, and 27 months of age were separated by gradient gel electrophoresis. Hypertrophy was assessed by indexing regional heart mass to tibial length From 9 through 27 months of age, hypertrophy was 19% and 77% in the left ventricle and left atrial appendage, respectively. There was no significant hypertrophy in either the right ventricular free wall or the right atrial appendage. The proportion of alpha-myosin heavy chain ranged from 86 +/- 1.3% (mean +/- SE) in the right ventricular free wall to 62 +/- 5.8% in left ventricular papillary muscle of 9-month-old rats. In 27-month-old rats, it ranged from 59 +/- 2.7% in the right ventricular free wall to 20 +/- 3.1% in the left ventricular papillary muscle. There was a marked age-associated decrease in the proportion of alpha-myosin heavy chain overall (p <.001) that did not differ significantly among the regions studied (p = .109). These results suggest that the effects of advancing age on the cardiac MHC pattern are independent of age-associated hypertrophy.     

Age-associated increases in the activity of multiple caspases in Fisher 344 rat organs

As organisms age, an increase in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling positive cells has been observed in a variety of tissues and cell types. However, whether this represents the increase of apoptosis has not been validated on molecular level. In this study we examined the endogenous activity of caspases that are known to be responsible for the execution of caspase-dependent apoptosis as a function of age in rat liver, lung, and spleen. We demonstrate that the extent of apoptosis in rat liver increases late during the aging process (i.e. 23-27 month) as indicated by the activation of executioner caspases-3, -6, and -7. We also found that the activity of caspase-3, -6, and -7 increased drastically in rat lung and spleen at late stages of aging. Despite reports that the level of Fas mRNA increases with age in rat liver and that Fas system regulates liver homeostasis, we did not detect activation of caspase-8, a key mediator of Fas-induced apoptosis, in aged liver. We also observed increased activities of two caspases, caspase-2 and caspase-9, which are involved in mitochondrion-mediated apoptosis in livers isolated from old rats, and found that hepatocytes isolated from old animals (>23 month) are more sensitive to oxidative stress that targets the mitochondria compared to those isolated from young (6 month) animals. Lastly, we demonstrate that the level of cytochrome c is lower in liver from old animals, probably as a result of expeditious degradation following its release into cytosol. Collectively, our results demonstrate that aging is associated with an increase in the activity of multiple caspases, suggesting that the extent of apoptosis increases as organs age. In the case of rat liver, this increase in caspase activation is more likely associated with the mitochondrial (i.e. intrinsic) pathway rather than the Fas-mediated caspase-8 (extrinsic) pathway of apoptosis.     

Phosphorylation of connexin in functional regulation of the cardiac gap junction

In cardiac muscle, the gap junction contributes to electrical cell-to-cell coupling. This physiological function of the gap junction depends on the phosphorylation state of the connexin molecule, which comprises the gap junction channel. The effects of intracellular Ca²⁺ overload, acidosis, activation of protein kinase (PK) A, PKC and PKG on the phosphorylation and expression of connexin 43 (Cx43) were examined in animal hearts with reference to physiological function. Activation of PKA promotes cell-to-cell coupling due to augmentation of the PKA-mediated phosphorylation of Cx43, with a rise in the quantity of and an increase in the expression of Cx43. A rise in the ionic strength of Ca²⁺ and H⁺ impaired cell communication, with the inhibition of PKA-mediated Cx43 phosphorylation. Activation of PKC reduces the quantity and expression of Cx43 despite augmentation of PKC-mediated phosphorylation of the protein. The effects of PKG activation are similar to those of PKC activation. It is suggested that PKA activation upregulates and PKC activation downregulates Cx43. The role of connexin phosphorylation in the regulation of gap junction function is discussed.     

心脏连接蛋白43羧基末端相互作用蛋白的酵母双杂交筛选

目的研究心脏连接蛋白43（Cx43）羧基末端与哪些心肌细胞内蛋白质存在相互作用。方法①通过PCR方法得到编码心脏Cx43羧基末端（AA235-382）的cDNA片段,并在其两端分别加上EcoRI和BamHI酶切位点,应用EcoRI/BamHI酶切PCR产物及pGBKT7空载体,凝胶分离后应用T4连接酶进行连接;②通过化学转化法将pG-BKT7-Cx43-CT转化酵母菌AH109;③通过“尿素/SDS”法从被转化的酵母菌中提取蛋白质;④应用抗C-myc抗体,通过Western blot方法检测”诱饵”蛋白的表达（即Gal4-BD-C-myc-Cx43-CT融合蛋白）;⑤检测”诱饵”蛋白自我激活报告基因与否后,将已被“诱饵“质粒转化的AH109与人心脏cDNA文库进行杂交,筛选阳性克隆分离阳性克隆中的cDNA,并测序。结果①诱饵载体测序结果证明pGBKT7中的“Gal4 DNA binding domain-C-myc”与Cx43的羧基末端在同一读框中;②“诱饵”质粒转化AH109酵母菌成功率100%;③从被转化的AH109中能提取到浓度满意的总蛋白;④Western blot能检测到特异性条带,其位置与Gal4-BD-C-myc-Cx43-CT的分子量相当;⑤“诱饵”蛋白不能自激活报告基因Ade2和Mel1,但可自激活His3,5mmol/L的3-AT可有效抑制“诱饵”蛋白本身自激活报告基因His3,以利于下一步的杂交筛选,筛选得到10个准阳性克隆。结论心脏连接蛋白43羧基末端能与心肌细胞中的多种蛋白质存在相互作用,这些蛋白质可能参与对间隙连接通道的功能调控。