黄芪总黄酮对病毒性心肌炎小鼠心律失常与内质网应激因子的影响

目的：研究黄芪总黄酮（TFA）对病毒性心肌炎小鼠心律失常与内质网应激及缝隙连接蛋白作用,明确TFA抗病毒性心肌炎合并心律失常作用机制。方法：36只雄性Balb/c小鼠分为正常对照组、病毒性心肌炎组和TFA组（n=12）,病毒性心肌炎组腹腔内无菌注射含0.1 ml/d 10-950 TCID柯萨奇B3病毒（CVB3）,注射3 d制备Balb/c小鼠病毒性心肌炎模型,TFA组给予CVB3同时尾静脉注射0.1 ml TFA （20 mg/L）,共7 d。实验结束后心电图检测心律失常发生率后处死小鼠,取心脏行HE染色,观察心肌病理改变,Western blot检测各组小鼠心肌细胞葡萄糖调节蛋白78（GRP78）、内质网应激信号通路因子激活转灵因子4（ATF4）及缝隙连接蛋白（Cx43）表达。结果：与正常组比较,病毒性心肌炎组GRP78与ATF4的表达显著升高（P<0.01）,Cx43表达明显下降（P<0.01）;与病毒性心肌炎组比较,TFA组小鼠心肌细胞GRP78与内质网应激信号通路因子ATF4表达明显减少（P<0.01）,Cx43表达明显增多（P<0.01）。结论：TFA抗心律失常作用可能与缓解内质网应激及增加Cx43表达有关。     

Stretch-induced regional mechanoelectric dispersion and arrhythmia in the right ventricle of anesthetized lambs

Regional mechanical and electrophysiological changes accompany most ventricular arrhythmias and, it has been suggested, by mechanoelectric feedback. We hypothesized that an intervention producing regional mechanical dispersion was associated with regional, proarrhythmic electrical dispersion and studied the regional mechanoelectric feedback in the right ventricle (RV) of anesthetized lambs. Ten lambs were deeply anesthetized, and their hearts were exposed. Three tripodal devices, each incorporating three monophasic action potential electrodes and an integrated strain-gauge system, were placed on the RV apex outflow and inflow regions. Measurements were made before, during, and after 10-s pulmonary arterial occlusion. Pulmonary arterial occlusion increased RV pressure and overall regional segment length. Length excursion became out of phase with RV pressure beats immediately after occlusion, and the strain patterns were different in the three regions at the peak of occlusion. The occlusion resulted in different alterations in regional monophasic action potential morphology, including reduction in monophasic action potential amplitude and duration by different amounts and early afterdepolarizations that were unevenly distributed in the monophasic action potential recordings. This was associated with dispersion of repolarization and recovery time. The combination of electromechanical events precipitated a variety of arrhythmias. Acute RV distension is proarrhythmic, possibly through a causal relationship among mechanically induced afterdepolarizations, dispersion (heterogeneity) of mechanical strain, and dispersion of electrical recovery. The relationship among the different wall motions, the dispersion of repolarization, and arrhythmia underscored mechanoelectric feedback as an important part of arrhythmogenesis in pulmonary embolism and commotio cordis.     

Cell Junctions in the Specialized Conduction System of the Heart

Abstract

Anchoring cell junctions are integral in maintaining electro-mechanical coupling of ventricular working cardiomyocytes; however, their role in cardiomyocytes of the cardiac conduction system (CCS) remains less clear. Recent studies in genetic mouse models and humans highlight the appearance of these cell junctions alongside gap junctions in the CCS and also show that defects in these structures and their components are associated with conduction impairments in the CCS. Here we outline current evidence supporting an integral relationship between anchoring and gap junctions in the CCS. Specifically we focus on (1) molecular and ultrastructural evidence for cell–cell junctions in specialized cardiomyocytes of the CCS, (2) genetic mouse models specifically targeting cell–cell junction components in the heart which exhibit CCS conduction defects and (3) human clinical studies from patients with cell–cell junction-based diseases that exhibit CCS electrophysiological defects.     

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.     

白介素11拮抗剂对实验性小鼠肺纤维化的作用

目的:观察拮抗白介素11(IL-11)对博来霉素(BLM)诱导的实验性小鼠肺纤维化的作用.方法:将120只雄性C57BL/6小鼠随机分为正常对照组、IL-11拮抗剂组、BLM组和BLM+IL-11拮抗剂组(每组各30只).BLM组和BLM+IL-11拮抗剂组小鼠一次性气管注射BLM(1.5 mg/kg)诱导肺纤维化.于...     

Myocardial fibrosis and right ventricular function of heterotopically transplanted hearts in rats treated with cyclosporin

The aim was to determine whether treatment of rats with cyclosporin A (CsA) leads to deleterious side effects on heterotopically iso- or allotransplanted hearts when compared with recipient native in situ hearts. Four experimental groups were employed: inbred (Lewis) rats receiving either no immunosuppression or CsA at a dose of 15 mg.kg^–1 per day for 7 days after surgery, and outbred (Wistar) rats receiving CsA at the same daily dose for either 7 or 21 days. One month following surgery, the mass of all transplanted hearts decreased and resulting atrophy was associated with relative myocardial fibrosis. Treatment with CsA significantly increased the concentration and content of collagen in the right and left ventricles of all transplanted and recipient hearts. No appreciable difference was observed between corresponding hearts of inbred and outbred groups receiving the identical dose of CsA, and between hearts in outbred groups treated for either 7 or 21 days. No signs of right ventricular mechanical dysfunction, as assessed on the isolated perfused working preparation, were observed after CsA treatment in both transplanted and recipient hearts. The maximal steady state developed pressure (RVDevP) and the rate of its development [(+dP/dt)_max] were slightly higher in transplants than in the corresponding recipients, and in CsA-treated versus untreated hearts, while the index of contractile state [(+dP/dt)/P] was similar in all groups. The data suggest that treatment of rats with CsA can induce a similar degree of fibrosis both in heterotopic cardiac transplants and in recipient native hearts without impairment of their contractile performance.     

Pannexin通道蛋白功能研究概述

Pannexin基因是2000年发现的缝隙连接蛋白家族新成员。目前研究表明,Pannexin蛋白(Px)可以在细胞膜上组成半通道或在细胞间形成缝隙连接通道,介导细胞内ATP释放、细胞间钙波传递、味觉感受、血管血流调节以及免疫应答等多种生理功能。在病理状态下,Px参与炎症、肿瘤、脑缺血、癫痫以及心衰等重大疾病发生、发展。随着Pannexin研究领域的深入,其生理病理状态下的更多重大功能将被阐释。     

Effects of stochastic channel gating and distribution on the cardiac action potential

Ion channels exhibit stochastic conformational changes determining their gating behavior. In addition, the process of protein turnover leads to a natural variability of the number of membrane and gap junctional channels. Nevertheless, in computational models, these two aspects are scarcely considered and their impacts are largely unknown. We investigated the effects of stochastic current fluctuations and channel distributions on action potential duration (APD), intercellular conduction delays (ICDs) and conduction blocks using a modified ventricular cell model (Rudy et al.) with Markovian formulations of the principal ion currents (to simulate their stochastic open-close gating behavior) and with channel counts drawn from Poisson distributions (to simulate their natural variability). In single cells, APD variability (coefficient of variation: 1.6% at BCL=1000 ms) was essentially caused by stochastic channel gating of I_Ks, persistent I_Na and I_Ca,L. In cell strands, ICD variability induced by stochastic channel gating and Poissonian channel distributions was low under normal conditions. Nonetheless, at low intercellular coupling levels, Poissonian gap junctional channel distribution resulted in a large ICD variability (coefficient of variation >20%), highly heterogeneous conduction patterns and conduction blocks. Therefore, the stochastic behavior of current fluctuations and channel distributions can contribute to the heterogeneity of conduction patterns and to conduction block, as observed previously in experiments in cardiac tissue with altered intercellular coupling.     

Expression of a dominant negative mutant innexin in identified neurons and glial cells reveals selective interactions among gap junctional proteins

Neurons and glia of the medicinal leech CNS express different subsets of the 21 innexin genes encoded in its genome. We report here that the punctal distributions of fluorescently tagged innexin transgenes varies in a stereotypical pattern depending on the innexin expressed. Furthermore, whereas certain innexins colocalize extensively (INX1 and INX14), others do not (e.g., INX1 and INX2 or INX6). We then demonstrate that the mutation of a highly conserved proline residue in the second transmembrane domain of innexins creates a gap junction protein with dominant negative properties. Coexpressing the mutated INX1 gene with its wild type blocks the formation of fluorescent puncta and decouples the expressing neuron from its normal gap junction‐coupled network of cells. Similarly, expression of an INX2 mutant transgene (a glial cell innexin), blocks endogenous INX2 puncta and wild‐type transgene puncta, and decouples the glial cell from the other glial cells in the ganglion. We show in cell culture with dye‐uptake and plasma membrane labeling experiments that the mutant innexin transgene is not expressed on the cell membrane but instead appears to accumulate in the cell's perinuclear region. Lastly, we use these mutant innexin transgenes to show that the INX1 mutant transgene blocks not only INX1 puncta formation, but also puncta of INX14, with which INX1 usually colocalizes. By contrast, the formation of INX6 puncta was unaffected by the INX1 mutant. Together, these experiments suggest that leech innexins can selectively interact with one another to form gap junction plaques, which are heterogeneously located in cellular arbors. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 571–586, 2013     

Atrial natriuretic factor in the impulse-conduction system of rat cardiac ventricles

Summary A complex network of atrial natriuretic factor-producing cells has been delineated by biochemical and morphological techniques in the rat ventricular myocardium. The chordae tendineae spuriae (CTS; false tendons) contain ANF mRNA and the ANF propeptide (Asn 1-Tyr 126) as assessed by Northern blot analysis, high-pressure liquid chromatography and immunohisto- and -cytochemistry, using three different affinity-purified antibodies: monoclonal and polyclonal antibodies against C-terminal ANF (Arg 101-Tyr 126) and polyclonal antibodies against N-terminal ANF (Asp 11-Ala 37). Two types of cells harboring ANF-containing secretory granules constitute the CTS: the majority (Purkinje type I) have ultrastructural similarities with both atrial and classical Purkinje fibers. Purkinje type-II fibers resemble working ventricular cardiocytes. Both cell types harbor a large paranuclear Golgi complex. The subendocardial Purkinje network is also made up of these two cell types. In this location, Purkinje type-I fibers form cable-like structures while Purkinje type-II fibers are either located beneath the former or abut directly on the endocardium. The latter are not separated from adjacent working ventricular cardiocytes by connective tissue septa. Coronary arteries and arterioles, as in birds, are surrounded by a cushion of Purkinje type-II fibers which blend with the surrounding myocardium. These results indicate that, in the rat, the entire intraventricular conduction system is constituted of endocrine cells producing ANF.Supported by a Medical Research Council of Canada Group Grant to the Multidisciplinary Research Group on Hypertension, by the National Research Council of Canada, the Pfizer Company (England), Bio-Méga Inc. and the Canadian Heart Foundation     

Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins

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Molecular mobility in the gap regions of type 1 collagen fibrils

Recent studies of the structure of Type I collagen fibrils (Piez and Trus,Biosci. Rep. 1:801–810, 1981; Fraser, MacRae, Miller and Suzuki,J. Mol. Biol. 167:497–521, 1983) suggest that the segments of the collagen molecule which comprise the gap region are more mobile than those which comprise the overlap region. We have analyzed the distribution of amino acid residues and triplet types between the two regions, and find significantly non-uniform distributions for Ala, Gln, His, Hyp, Leu, Phe, and Tyr, and for triplets containing two imino acid residues. Taken together with the lower packing density in the gap region these observations provide a basis for understanding the greater mobility of the molecular segments in the gap region. In addition, we have examined the linear distribution of residue types in the two regions and also the hydropathy profile (Kyte and Doolittle,J. Mol. Biol. 157: 105–113, 1982). These reveal a segment of the gap region comprising helical residues 165–173, 399–407, 633–641 and 867–975 which has the highest hydropathy index, is devoid of charged residues, and contains very high proportions of Ala, Hyp and Phe.     

In vivo effect of growth hormone on the expression of connexin-43 in bovine ovarian follicles

This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis.     

Protein remodeling of the heart ventricles in hereditary hypertriglyceridemic rat: effect of ace-inhibition

The aim of this study was to determine whether protein remodeling of the heart ventricles and remodeling of the aorta were present in hereditary hypertriglyceridemic (hHTG) rats and whether treatment with the angiotensin-converting enzyme inhibitor, captopril could prevent these alterations. Three groups of rats were investigated in a four week experiment control Wistar /C/rats, hHTg rats, hHTg rats given captopril (100 mg/kg/day) (hHTg + CAP). In the hHTg group, the increased systolic blood pressure (SBP) was associated with hypertrophy of the LV and RV. Protein profile analysis revealed an enhancement of metabolic protein concentration in both ventricles. The concentration of total collagenous proteins was not changed in either ventricles. However, alterations in composition of cardiac collagen were detected, characterized by higher concentration of hydroxyproline in pepsin-insoluble fraction and lower concentration of hydroxyproline in pepsin soluble faction in the LV. Hypertrophy of aorta, associated with the reduction of nitric oxide dependent relaxation, was also present in hHTG rats. Captopril normalized SBP, reduced left ventricular hypertrophy (LVH), diminished metabolic protein concentration in both ventricles, and improved NO-dependent relaxation of the aorta. Furthermore, captopril partially reversed alterations in hydroxyproline concentration in soluble and insoluble collagenous fractions of the LV. We conclude that hypertrophy of both ventricles and the aorta are present in hHTG rats, along with protein remodeling of both ventricles. Captopril partially prevented left ventricular hypertrophy development and protein remodeling of the myocardium.     

Conditional Gene Targeting of Connexin43: Exploring the Consequences of Gap Junction Remodeling in the Heart

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.     

Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40

Cells within the vascular wall are coupled by gap junctions, allowing for direct intercellular transfer of low molecular weight molecules. Although gap junctions are believed to be important for vascular development and function, their precise roles are not well understood. Mice lacking either connexin37 (Cx37) or connexin40 (Cx40), the predominant gap junction proteins present in vascular endothelium, are viable and exhibit phenotypes that are largely non-blood vessel related. Since Cx37 and Cx40 are coexpressed in endothelial cells and could overlap functionally, some roles of junctional communication may only be revealed by the elimination of both connexins. In this study, we interbreed Cx37 and Cx40 knockout mice to generate Cx37-/- Cx40-/- animals and show that they display severe vascular abnormalities and die perinatally. Cx37-/- Cx40-/- animals exhibit localized hemorrhages in skin, testis, gastrointestinal tissues, and lungs, with pronounced blood vessel dilatation and congestion occurring in some areas. Vascular anomalies were particularly striking in testis and intestine. In testis, abnormal vascular channels were present, with these channels coalescing into a cavernous, endothelium-lined blood pool resembling a hemangioma. These results provide evidence of a critical role for endothelial gap junction-mediated communication in the development and/or functional maintenance of segments of the mouse vasculature.     

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.     

外源性精胺对糖尿病肾病小鼠肾纤维化的影响及其机制

目的: 观察外源性精胺对糖尿病肾病(DN)肾纤维化的保护作用,并探讨其机制。方法: 24 只雄性 C57 小鼠随机分为正常组(Control)、糖尿病组(T1D)和精胺预处理组(T1D+Sp,每组 n=8)。一次性注射 STZ(60 mg/kg)复制 1 型糖尿病小鼠模型,精胺预处理组在 STZ 注射前两周每天腹腔注射精胺(Sp,5 mg/(kg·d)),随后隔天注射精胺,第 12 周处死小鼠,检测血清肌酐、尿素氮判断肾功能变化,HE、PAS 和 Masson 染色观察肾组织损伤和纤维化水平。Western blot 法检测小鼠肾组织中基质金属蛋白酶(MMP-2、MMP-9)、IV型胶原(Coll-IV)蛋白的表达。结果: 与 Control 相比,T1D 组血糖(5.67±0.22 vs 28.40±0.57 mmol/L)、肌酐(14.33±1.22 vs 30.67±4.73 μmol/L)、尿素氮(6.93±4.94 vs 22.00±1.04 mmol/L)明显升高(P＜0.05),肾组织基底膜增厚,胶原含量明显增加,MMP-2、MMP-9 和 Coll-IV 蛋白表达均升高(分别为 0.57±0.07 vs 1.06±0.20、47.00±0.04 vs 1.29±0.09和0.42±0.16 vs 0.95±0.18,P＜0.05),精胺预处理明显减轻上述变化。结论: 外源性精胺预处理通过调节 MMPs 与胶原的平衡减轻 DN 小鼠的肾纤维化。     

Schistosoma mekongi and Schistosoma japonicum: Differences in the distribution of eggs in the viscera of mice

The difference in the distribution of Schistosoma eggs in the viscera has not been clearly elucidated in the two closely related species Schistosoma japonicum and Schistosoma mekongi. In this study, we quantitatively compared the distribution of eggs in mice infected with the two species. In S. mekongi-infected mice, 56.6% to 69.4% of total eggs were found in the distal small intestine 9 to 15 weeks after infection, while in S. japonicum-infected mice, 48.8% to 71.8% of eggs were found in the proximal small intestine during the same period. There were significantly more eggs in the liver in mice infected with S. japonicum than in those infected with S. mekongi. The number of adult worms recovered did not differ between the two species during the study period. The total number of eggs laid in the tissues also did not differ between the two species at 12 to 15 weeks postinfection, but in the earlier period the total number of eggs was significantly fewer in S. mekongi-infected than in S. japonicum-infected mice, suggesting the delayed maturation of the former compared with the latter. These results clearly show that S. japonicum and S. mekongi exhibit different oviposition behavior in their hosts.