ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.     

The role of beta 1 integrin in spreading and myofibrillogenesis in neonatal rat cardiomyocytes in vitro.

The influence of the extracellular matrix (ECM) on cell behavior, myofibrillogenesis and cytoarchitecture was investigated in neonatal rat cardiac myocytes in vitro. Cell behavior was examined by analyzing cell spreading on different ECM components under a variety of experimental conditions. Area measurements were made on digitized images of cells grown for various time intervals on fibronectin (FN), laminin (LN), collagens I and III (C I+III), plastic, and bovine serum albumin (BSA). The amount of spreading was varied on the different matrices and was maximal on FN greater than LN greater than C I+III greater than plastic greater than BSA. Addition of anti-beta 1 integrin antibodies to myocytes cultured on FN, LN and C I+III blocked spreading outward on the substrates and altered normal myofibrillogenesis, especially on LN. Concomitantly, the integrin antibodies induced the formation of giant pseudopodial processes which protruded upward from the substrates. These pseudopods contained actin polygonal networks which exhibited a regular geometrical configuration. Effects of the ECM on cytoarchitecture was examined by analyzing the temporal and spatial patterns of fluorescence and immunogold labeling of cytoskeletal and integrin proteins as myocytes spread in culture. The first indication of sarcomeric patterns was the appearance at 4 hours of striations formed by lateral alignment of alpha-actinin aggregates into Z bands. At later times, vinculin at 8 hours and beta 1 integrin at 22 hours became co-localized with alpha-actinin at the Z bands and focal adhesions. These data indicate that ECM components influence myocyte spreading and that myofibril assembly and/or stability is associated with ECM-integrin-cytoskeleton associations.     

Studies of hamster cardiac myofibrillogenesis in vivo with antibodies to spectrin, desmin, and alpha-actinin

The spectrins are a family of cytoskeletal-membrane proteins that have generated much interest in the past decade. In the present study, we utilized immunohistochemical, morphological, and electrophoretic techniques to assess the possible function(s) of spectrin in mammalian cardiac tissue during development. Antibodies generated against alpha-actinin and desmin were also employed to identify myofibrils and intermediate filaments in relation to changes in the distribution of spectrin. Spectrin is localized along the sarcolemma of pre-myofibrillar hamster cardiac myocytes (day 8, postcoitum) and remains associated with the cell membrane throughout development. The staining pattern is somewhat diffuse at first, but eventually the cell margin becomes clearly defined by day 13 postcoitum. A second, more profound change in the distribution of spectrin occurs during the newborn stage, when spectrin begins to appear in the sarcoplasm. It appears as regularly spaced invaginations that are diffuse at first, eventually attaining a position around the Z-bands of adult muscle. The change in the distribution of spectrin coincides temporally with the appearance of T-tubules, which are sarcolemmal invaginations that reside at the Z-bands of adult heart. Thus, spectrin may act as a guidance mechanism for the proper positioning of T-tubules around the Z-discs of mammalian cardiac tissue. Although spectrin does not appear to interact directly with early myofibrils it may assist in the proper alignment of T-tubules and, in doing so, act to stabilize the entire contractile apparatus by enveloping it and attaching it to the sarcolemma.     

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of micro filaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cyto-skeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function. This work was supported by grants from the Medical Research Council of Canada     

Immunofluorescent localization of myosin, alpha-actinin and tropomyosin in odontoblast microfilament bundles of the rat incisor

The localization of alpha-actinin, tropomyosin, myosin and actin in odontoblasts was examined by fluorescence microscopy using well characterized antibodies and rhodamine-phalloidin. All the reagents labeled the distal end of the cell body in the form of an oval ring with a preferential axis along the tooth axis. This ring was often interrupted. In conventional electron microscopy, microfilament bundles with periodical dense spots were running along the tooth axis at the level of the distal end of the cell body. The periodicity was about 0.6-1.0 microns. It may be possible that this dynamic structure functions to keep odontoblasts in a layer by contracting in an isometric form.     

An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment

The LIM domain protein zyxin is a component of adherens type junctions, stress fibers, and highly dynamic membrane areas and appears to be involved in microfilament organization. Chicken zyxin and its human counterpart display less than 60% sequence identity, raising concern about their functional identity. Here, we demonstrate that human zyxin, like the avian protein, specifically interacts with alpha-actinin. Furthermore, we map the interaction site to a motif of approximately 22 amino acids, present in the N-terminal domain of human zyxin. This motif is both necessary and sufficient for alpha-actinin binding, whereas a downstream region, which is related in sequence, appears to be dispensable. A synthetic peptide comprising human zyxin residues 21-42 specifically binds to alpha-actinin in solid phase binding assays. In contrast to full-length zyxin, constructs lacking this motif do not interact with alpha-actinin in blot overlays and fail to recruit alpha-actinin in living cells. When zyxin lacking the alpha-actinin binding site is expressed as a fusion protein with green fluorescent protein, association of the recombinant protein with stress fibers is abolished, and targeting to focal adhesions is grossly impaired. Our results suggest a crucial role for the alpha-actinin-zyxin interaction in subcellular zyxin localization and microfilament organization.     

Contractile proteins in podocytes: immunocytochemical localization of actin and alpha-actinin in normal and nephrotic rat kidneys.

Actin and alpha-actinin immunoreactive sites have been localized at the electron microscope level by the protein A-gold immunocytochemical technique in podocytes of normal and nephrotic rat renal tissues. In normal renal glomeruli, fibrillar networks located in the core of foot processes or bundles of microfilaments interconnecting them were found to be labelled for these two cytoskeletal proteins. On the other hand, in nephrotic renal glomeruli, concomitant with the loss of podocytic foot processes a reorganization of the podocytic cytoskeleton and a concentration of some of its elements into thick uniform bands was observed. Actin and alpha-actinin were revealed in these bands. Control experiments confirmed the specificity of the labelling obtained. Our results suggest that normal podocytes contain an actin-based contractile system that might contribute to the maintenance of the particular cell shape of these cells and that the rearrangement of the podocytic cytoskeleton occurring in the nephrotic syndrome might account for the changes in the foot processes and contribute to the alteration in glomerular function.     

Myofibril assembly visualized by imaging N-RAP, alpha-actinin, and actin in living cardiomyocytes

N-RAP is a striated muscle-specific scaffolding protein that organizes α-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either α-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of α-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The α-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the α-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before α-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.     

Developmental changes of calcium transients and contractility during the cultivation of rat neonatal cardiomyocytes

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.This work was supported in part by NIH grant GM-48595 and by a Grant-In-Aid from the American Heart Association, Missouri Affiliate (92-10517).     

Expression of glutamate transporters in rat cardiomyocytes and their localization in the T-tubular system.

Glutamate and aspartate play important roles in the intermediary metabolism of the myocardium and have been shown to improve cardiac recovery after hypoxia or ischemia. Limited data are available about the expression of glutamate transporters that are involved in the uptake of glutamate and aspartate in cardiomyocytes. In this study, non-radioactive in situ hybridization (ISH) using complementary RNA probes was applied to detect the glutamate transporters GLT1 variant (GLT1v) and EAAC1 mRNA in rat cardiomyocytes. The transporter proteins were demonstrated by Western blotting and immunocytochemistry using affinity-purified antibodies against transporter peptides. ISH and immunocytochemistry showed that both glutamate transporters are coexpressed in cardiomyocytes. The ISH labeling indicates the distribution of transporter mRNA throughout the cytoplasm of cardiomyocytes. GLT1v and EAAC1 proteins, which showed in Western blots a molecular mass of approximately 60 kD, are strongly enriched and colocalized in the transverse (T)-tubular system of cardiomyocytes. These results may indicate that glutamate/aspartate uptake into cardiomyocytes could be mediated by the high-affinity transporters GLT1v and EAAC1. A high efficiency of glutamate/aspartate transport into cardiomyocytes could be achieved by their localization in the T-tubular system, which consists of tubular invaginations of the sarcolemma extending deep into the cell.     

Immunoelectron microscopic localization of alpha-actinin and actin in embryonic hamster heart cells

Myofibrillogenesis in developing cardiac cells of the Syrian hamster from early embryonic stages through newborn was studied by electron microscopy, immunofluorescence microscopy and immunoelectron microscopy. alpha-Actinin and actin were localized at light and electron microscopic levels in embryonic heart cells which had been fixed in a periodate-lysine-paraformaldehyde or a glutaraldehyde-formaldehyde mixture, and embedded in Lowicryl K4M. Indirect staining methods were used for immunofluorescence staining of thick sections and immunoferritin staining of thin sections. The earliest evidence of myofibrillogenesis in embryonic myocardial cells was the presence of many randomly arranged thin (6 nm) filaments and a few scattered thick filaments (15 nm) near the plasma membrane. alpha-Actinin was detected in a semi-continuous, diffuse layer in some portions of the cell just beneath the plasma membrane in association with the filamentous collections. Later in development, alpha-actinin coalesced into Z-plaques at the membrane as the filaments arranged into parallel arrays. Actin was localized in the thin filaments as expected. In later stages of development, alpha-actinin was observed at the Z-lines and intercalated discs of the mature myofibrils while actin was localized at both the I-band and Z-line. Our results suggest that myofibrillogenesis is initiated at the plasma membrane and that Z-plaques are precursors of myofibrillar Z-bands and may serve as organizing centers for myofibrillogenesis in developing cardiomyocytes.     

Physico-chemical properties of rat and dog cardiac alpha-actinin

alpha-Actinin exists in several polymorphic forms which appear to be characteristic of the muscle type from which it is isolated. In order to determine the possible physiological role of this structural protein in cardiac muscle, we describe and compare here the physico-chemical properties of cardiac alpha-actinin from two different mammalian species, rat (fast contracting muscle) and dog (slow contracting muscle). Purification of cardiac alpha-actinin was achieved by chromatography on DEAE-cellulose and hydroxyapatite columns. The alpha-actinins isolated were different in their electrophoretic mobility (SDS-polyacrylamide gel electrophoresis), molecular size and alpha-helical content. However, their shape as revealed by electron microscopy and their activating effect on Mg2+-ATPase activity of actomyosin appear to be similar. These studies suggest that the rat and dog cardiac alpha-actinin are structurally different but functionally similar proteins.     

Ginseng: Cardiotonic in adult rat cardiomyocytes, cardiotoxic in neonatal rat cardiomyocytes

Ginsengs are widely used to improve cardiac health and circulation. Loosely termed as ginsengs, Asian (Panax), Siberian and Ashwagandha (Indian Ginseng) Indian ginsengs are prepared from different plants. We tested the popular belief of cardiotonic effects of ginsengs using both neonatal and adult rat cardiomyocytes, comparing extracts from the three ginsengs. Addition of 10% v/v of extract (100 microl of extract/ml of culture medium) of each of the ginsengs resulted in a rapid (<10 s) cessation of beating in neonatal cardiomyocytes due to calcium overload, while sequential dilutions revealed that treatment with a low dose (0.01% v/v, 0.1 microl/ml of the medium) resulted in constant, regular beats (transients), and a slight elevation of diastolic calcium without overload. Addition of extracts to sparking, calcium-tolerant adult cardiomyocytes resulted in initiation of calcium transients, and adult cells were able to tolerate exposure to high concentrations of extract. Cardiotonic effects in adult cells (cardiotoxicity in neonatal cells) were most profound with Asian ginseng (2.6 times that of Siberian ginseng, 1.6 times that of Indian ginseng) probably due to the active ingredients (ginsenosides in Asian, eleutherosides in Siberian and withanolides in Indian) being structurally different. We conclude that fully developed cardiomyocytes are able to accommodate higher doses of ginseng than neonatal cells, and that the effects of ginseng on newly formed, developing myocytes, could be extremely deleterious to the fetus. However, for adults, ginseng might well be a 'tonic' in its ability to increase beating and intramyocytic calcium levels.     

Changes in GM1 ganglioside content and localization in cholestatic rat liver

(Glyco)sphingolipids (GSL) are believed to protect the cell against harmful environmental factors by increasing the rigidity of plasma membrane. Marked decrease of membrane fluidity in cholestatic hepatocytes was described but the role of GSL therein has not been investigated so far. In this study, localization in hepatocytes of a representative of GSL, the GM1 ganglioside, was compared between of rats with cholestasis induced by 17α-ethinylestradiol (EE) and vehicle propanediol treated or untreated animals. GM1 was monitored by histochemical reaction employing cholera toxin B-subunit. Our findings in normal rat liver tissue showed that GM1 was localized in sinusoidal and canalicular hepatocyte membranes in both peripheral and intermediate zones of the hepatic lobules, and was nearly absent in central zones. On the contrary, in EE-treated animals GM1 was also expressed in central lobular zones. Moreover, detailed densitometry analysis at high magnification showed greater difference of GM1 expression between sinusoidal surface areas and areas of adjacent cytoplasm, caused as well by increased sinusoidal staining in central lobular zone as by decreased staining in cytoplasm in peripheral zone. These differences correlated with serum bile acids as documented by linear regression analyses. Both GM1 content and mRNA corresponding to GM1-synthase remained unchanged in livers; the enhanced expression of GM1 at sinusoidal membrane thus seems to be due to re-distribution of cellular GM1 at limited biosynthesis and could be responsible for protection of hepatocytes against harmful effects of bile acids accumulated during cholestasis.     

Functional state of rat cardiomyocytes and blood antioxidant system under psycho-emotional stress

We studied the functionality of the antioxidant system in laboratory rat cardiomyocytes and blood under psycho-emotional stress. It was found that 40-day isolation and violation of diurnal cycle among the animals were accompanied by the intensification of lipid peroxidation process and marked with a reduced activity of antioxidant system enzymes, such as catalase and superoxide dismutase activity. The results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiomyocytes, which was further strengthened by the fact that the activity of the enzymes involved in ATP synthesis in mitochondria was reduced. Based on the results, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases.     

Nuclear localization of aldolase A in pig cardiomyocytes

The subcellular localization of the muscle aldolase (aldolase A) in cardiomyocytes was determined immunocytochemically by light and electron microscopy. The enzyme was localized in the cytoplasm and also in cardiomyocyte nuclei. Inside the nuclei it was preferentially localized in the heterochromatin region. The nuclear localization was confirmed by the measurement of aldolase activity in subcellular fractions of a heart muscle, and in isolated nuclei of cardiomyocytes. There was no detectable aldolase activity in isolated cardiomyocyte nuclei fractions if the fraction was not preincubated with a solution containing Triton X-100 and KCl. The calculated concentration of aldolase in the nucleus was about 0.6 micro M. This paper is the first report on the localization of aldolase A inside cardiomyocyte nuclei.     

机械作业下土壤理化性质和生态因子的变化

通过 3年对黄淮海农业机械化推广的研究 ,探讨了机械综合作业对旱作农业的影响 .结果表明 ,机械化秸秆还田、化肥深施、土壤深松和免耕覆盖等综合技术的合理配套实施 ,可以改善土壤理化性状 ,具有明显的生物学效应 .与传统作业相比 ,机械综合作业使土壤容重减少 0 .0 8g·cm-3 ,有机质含量增加12 % ,水分利用率提高 10 .1%～ 13.6 % ,小麦玉米产量增加 12 18kg·hm-2 .     

Tissue transglutaminase 2 promotes apoptosis of rat neonatal cardiomyocytes under oxidative stress

The role of tissue transglutaminase 2 (TG2) in cardiac myocyte apoptosis under oxidative stress induced by ischemic injury remains unclear. Here, we investigated the effects of TG2 on apoptosis of cardiomyocytes under oxidative stress. Ectopic expression of TG2 increased caspase-3 activity and calcium overload in cardiomyocytes. Expression levels of TG2 were significantly increased in H₂O₂-treated cardiomyocytes. Caspase-3 activity assay demonstrated its considerable correlation with TG2 expression, which supported that caspase-3 inhibitor inhibited the apoptosis induced by the ectopic overexpression of TG2. In addition, the other apoptotic signals, such as caspase-8, cytochrome c, and Bax, were increased dependent with TG2 expression in H₂O₂-treated cardiomyocytes. These results indicated that apoptotic signals had a positive correlation with TG2 expression. The decreased expression of phospholipase C (PLC)-δ1 and phospho-PKC in H₂O₂-treated cardiomyocytes were rescued by TG2 silencing. Together, our data strongly suggest that oxidative stress up-regulates TG2 expression in cardiomyocytes, leading to apoptosis.     

Tissue transglutaminase 2 promotes apoptosis of rat neonatal cardiomyocytes under oxidative stress

The role of tissue transglutaminase 2 (TG2) in cardiac myocyte apoptosis under oxidative stress induced by ischemic injury remains unclear. Here, we investigated the effects of TG2 on apoptosis of cardiomyocytes under oxidative stress. Ectopic expression of TG2 increased caspase-3 activity and calcium overload in cardiomyocytes. Expression levels of TG2 were significantly increased in H(2)O(2)-treated cardiomyocytes. Caspase-3 activity assay demonstrated its considerable correlation with TG2 expression, which supported that caspase-3 inhibitor inhibited the apoptosis induced by the ectopic overexpression of TG2. In addition, the other apoptotic signals, such as caspase-8, cytochrome c, and Bax, were increased dependent with TG2 expression in H(2)O(2)-treated cardiomyocytes. These results indicated that apoptotic signals had a positive correlation with TG2 expression. The decreased expression of phospholipase C (PLC)-δ1 and phospho-PKC in H(2)O(2)-treated cardiomyocytes were rescued by TG2 silencing. Together, our data strongly suggest that oxidative stress up-regulates TG2 expression in cardiomyocytes, leading to apoptosis.