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.     

Membrane fluidity changes are associated with the antiarrhythmic effects of docosahexaenoic acid in adult rat cardiomyocytes

Previous studies using neonatal rat cardiomyocytes have reported antiarrhythmic effects of long-chain polyunsaturated fatty acids (PUFAs). In this study, we examined the effects of the n-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on the spontaneous contractile activity and membrane fluidity of adult rat ventricular myocytes. Cardiomyocytes were induced to contract spontaneously by continuous superfusion of a solution containing the arrhythmogenic agents isoproterenol (a beta-adrenergic receptor agonist) or lysophosphatidylcholine. The percentage of cardiomyocytes displaying spontaneous contractions induced by isoproterenol when pretreated with the saturated fatty acid docosanoic acid was 48.1 +/- 7.7%; the percentage for cardiomyocytes pretreated with DHA was 7.1 +/- 2.4% (P < 0.01). DHA significantly prevented lysophosphatidylcholine-induced spontaneous contractions (17.7 +/- 6.5%) compared with treatment with the saturated fatty acid stearic acid (78.0 +/- 7.3%, P < 0.01). The membrane fluidizing agent benzyl alcohol also significantly prevented spontaneous contractions in cardiomyocytes. Membrane fluidity was determined by steady-state fluorescence anisotropy (r(ss)) using the fluorescent probe N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl) trimethyl-ammonium p-toluene-sulfonate (TMAP-DPH). DHA and benzyl alcohol dose-dependently decreased the r(ss); however, saturated fatty acids were without effect. These results suggest that the antiarrhythmic mechanisms of the n-3 PUFAs such as DHA may involve changes in membrane fluidity.     

培养成年大鼠心肌细胞存活的形态标志

目的：通过探寻增加培养成年大鼠心肌细胞存活率以及防止再分化的方法,揭示培养成年大鼠心肌细胞存活的形态标志。方法：采用Langendorff系统灌流心脏,胶原酶消化法分离成年大鼠心肌细胞,分3组进行细胞培养：①基础培养液＋凋亡抑制剂;②基础培养液＋5%胎牛血清;③基础培养液＋5%胎牛血清＋凋亡抑制剂。结果：①培养前3天杆状心肌细胞比例逐渐降低,无血清培养组比血清培养组降低程度大。培养前3天凋亡率逐渐升高,无血清培养组比血清培养组凋亡率高,加入凋亡抑制剂对凋亡率无影响。②有血清培养2~3天的成年大鼠心肌细胞闰盘部位伸出伪足,促使细胞贴壁生长;当培养至第6天时,细胞侧面也伸展出贴壁的伪足,细胞丧失杆状形态,横纹消失。而无血清培养的细胞无伪足生成,随着培养时间增加,细胞末端变圆钝,横纹变模糊。凋亡抑制剂对伪足形成率无影响。③培养存活的成年大鼠心肌细胞骨架重排,发生再分化。④血清培养组细胞胞内核间距离随着培养时间的增加而减小,无血清培养组则保持不变。结论：成年大鼠心肌细胞培养至第2~3天时,闰盘部位形成伪足是细胞存活的形态标志,加入血清是伪足形成的必要条件。     

Bioinformatic profiling of the transcriptional response of adult rat cardiomyocytes to distinct fatty acids

Diabetes mellitus, obesity, and dyslipidemia increase risk for cardiovascular disease, and expose the heart to high plasma fatty acid (FA) levels. Recent studies suggest that distinct FA species are cardiotoxic (e.g., palmitate), while others are cardioprotective (e.g., oleate), although the molecular mechanisms mediating these observations are unclear. The purpose of the present study was to investigate the differential effects of distinct FA species (varying carbon length and degree of saturation) on adult rat cardiomyocyte (ARC) gene expression. ARCs were initially challenged with 0.4 mM octanoate (8:0), palmitate (16:0), stearate (18:0), oleate (18:1), or linoleate (18:2) for 24 h. Microarray analysis revealed differential regulation of gene expression by the distinct FAs; the order regarding the number of genes whose expression was influenced by a specific FA was octanoate (1,188) > stearate (740) > palmitate (590) > oleate (83) > linoleate (65). In general, cardioprotective FAs (e.g., oleate) increased expression of genes promoting FA oxidation to a greater extent than cardiotoxic FAs (e.g., palmitate), whereas the latter induced markers of endoplasmic reticulum and oxidative stress. Subsequent RT-PCR analysis revealed distinct time- and concentration-dependent effects of these FA species, in a gene-specific manner. For example, stearate- and palmitate-mediated ucp3 induction tended to be transient (i.e., initial high induction, followed by subsequent repression), whereas oleate-mediated induction was sustained. These findings may provide insight into why diets high in unsaturated FAs (e.g., oleate) are cardioprotective, whereas diets rich in saturated FAs (e.g., palmitate) are not.     

Dual effect of heparin on cultured adult rat cardiomyocytes

Heparin has been widely reported to inhibit the growth of several cell types including neonatal rat cardiac myocyte (NRCM) but its effect on adult rat ventricular myocyte (ARVM) is unknown. To determine whether heparin is able to modulate ARVM protein synthesis capacity and if so which pathway is involved in this response, ARVM were cultured in presence or absence of 5% human serum and exposed to heparin (2-2,000 microg/ml) or its analogue xylan (0.5 and 50 microg/ml), and either the Ca(2+) chelator BAPTA/AM (10 microg/ml), or the calcineurin inhibitor FK506 (10 microg/ml), and heparinase I (0.1-10 U/ml) for 2 days. The protein synthesis (PS) was measured after 24 h incorporation of [14C]-Phenylalanine in ARVM. Independently of the serum presence, heparin and xylan altered PS in a bimodal dose-dependent manner. At high doses, heparin and xylan (2,000 and 50 microg/ml, respectively) either had no effect (without serum) or inhibited PS (with serum). In absence of serum, low doses of heparin or xylan (20 and 0.5 microg/ml, respectively) amplified the PS process in ARVM (2-fold, P < 0.05). FK506 inhibited the trophic response to 20 microg/ml heparin alone (-39%, P < 0.05). In presence of serum, the heparin induced-trophic effect, that was not significantly altered by FK506, was inhibited by BAPTA/AM (-32%, P < 0.05). Finally, heparinase I that increased PS in NRCM had no effect on ARVM growth. This study strongly suggests that heparin dose-dependently modulated PS in ARVM, this result being not observed in neonatal cells. Different mechanisms involving intracellular Ca(2+) play a role in the PS response of ARVM to low concentrations of heparin, the intracellular pathways depending on the presence of serum.     

Uncoupling protein 2 modulates cell viability in adult rat cardiomyocytes

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane proton carrier that uncouples ATP synthesis. The aim of this study was to determine whether UCP2 plays a role in survival of adult rat cardiac myocytes. We first studied the effects of UCP2 overexpression in vitro. Overexpression of UCP2 in primary cardiomyocytes led to a significant decline in ATP level and the development of acidosis but had no observable effect on cell survival. When cardiomyocytes were challenged with hypoxia-reoxygenation, cells overexpressing UCP2 survived significantly less compared with control. This finding was associated with upregulation of proapoptotic protein Bcl-2 and 19-kDa interacting protein 3 (BNIP3). Furthermore, UCP2 short interfering RNA prevented both the increase in cell death and BNIP3 expression. To examine the in vivo role of UCP2 in the heart, we used the Dahl salt-sensitive rat heart-failure model. Northern blot analysis revealed that UCP2 mRNA level was significantly upregulated in rat heart failure along with BNIP3 protein level. In conclusion, UCP2 increases sensitivity of adult rat cardiac myocytes to hypoxia-reoxygenation by way of ATP depletion and acidosis, which in turn causes accumulation of prodeath protein BNIP3.     

Effects of docosahexaenoic acid on calcium pathway in adult rat cardiomyocytes

In this study we examined the effect of polyunsaturated fatty acids (PUFAs), in particular of docosahexaenoic acid (DHA), on calcium homeostasis in isolated adult rat cardiomyocytes exposed to KCl, ET-1 and anoxia. Free [Ca(2+)](i) in rat cardiomyocytes was 135.7 +/- 0.5 nM. Exposure to 50 mM KCl or 100 nM ET-1 resulted in a rise in free [Ca(2+)](i) in freshly isolated cells (465.4 +/- 15.6 nM and 311.3 +/- 12.6 nM, respectively) and in cultured cells (450.8 +/- 14.8 nM and 323.5 +/- 14.8 nM respectively). An acute treatment (20 minutes) with 10 microM DHA significantly reduced the KCl- and ET-1-induced [Ca(2+)](i) increase (300.9 +/- 18.1 nM and 232.08 +/- 11.8 nM, respectively). This reduction was greater after chronic treatment with DHA (72 h; 257.7 +/- 13.08 nM and 192.18 +/- 9.8 nM, respectively). Rat cardiomyocytes exposed to a 20 minute superfusion with anoxic solution, obtained by replacing O(2) with N(2) in gas mixture, showed a massive increase in cytosolic calcium (1200.2 +/- 50.2 nM). Longer exposure to anoxia induced hypercontraction and later death of rat cardiomyocytes. Preincubation with DHA reduced the anoxic effect on [Ca(2+)](i) (498.4 +/- 7.3 nM in acute and 200.2 +/- 12.2 nM in chronic treatment). In anoxic conditions 50 mM KCl and 100 nM ET-1 produced extreme and unmeasurable increases of [Ca(2+)](i.) Preincubation for 20 minutes with DHA reduced this phenomenon (856.1 +/- 20.3 nM and 782.3 +/- 7.6 nM, respectively). This reduction is more evident after a chronic treatment with DHA (257.7 +/- 10.6 nM and 232.2 +/- 12.5 nM, respectively). We conclude that in rat cardiomyocytes KCl, ET-1 and anoxia interfered with intracellular calcium concentrations by either modifying calcium levels or impairing calcium homeostasis. Acute, and especially chronic, DHA administration markedly reduced the damage induced by calcium overload in those cells.     

Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes

Expression of alpha-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No alpha-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of alpha-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little alpha-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on alpha-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of alpha-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the alpha-sm-actin and alpha-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

PKC alpha-dependent regulation of the IGF1 receptor in adult and embryonic rat cardiomyocytes

In both, the adult rat ventricular cardiomyocytes and the embryonic rat heart cell line, H9c2, acute exposure to IGF1 resulted in activation of the IGF1 receptor’s internal tyrosine kinase, and this was completely blocked by the PKC alpha inhibitor, Gö6976. In addition, RNA interference using siRNA mediated gene silencing of PKC alpha–inhibited IGF1 receptor activity and blocked PKC alpha expression in H9c2 cells. Biochemical experiments demonstrate that PKC alpha is associated with the IGF1R (beta subunit) only after acute IGF1 exposure, and this may suggest that there is a direct interaction and possibly a PKC alpha phosphorylation site within the internal IGF1 receptor domain. The downstream effects of blocking PKC alpha activity by exposure to Gö6976 include inhibition of IGF1-stimuated PI3 kinase activity and reduced IGF1-stimulated c-fos expression in the adult cardiomyocytes. Previously, the laboratory has reported that IGF1 activates PKC alpha in adult rat cardiomyocytes, and that PKC alpha activity is required for IGF1-dependent Erk/Erk2 activity and protein synthesis. Here, it is shown that IGF1-dependent protein synthesis is completely blocked by PD98059, indicating that the Raf-Mek-Erk cascade is required for IGF1’s anabolic activity. Pretreatment with LY294002, a specific inhibitor of PI3 kinase, blocked IGF1-stimulated Erk1/Erk2 activity; therefore, PI3 kinase may also be required for IGF1-dependent protein synthesis. In H9c2 cells, coincubation with PMA lead to an increase in the rate of the IGF1 receptor activation, and this may further implicate a role for PKC in regulating the IGF1R. In conclusion, PKC alpha plays an essential role in the IGF1-signaling cascade, including the regulation of key signaling proteins involved in cell signaling and gene expression, and this may primarily be due to PKC alpha directly regulating the IGF1R.Both contributed equally to the experiments.     

Regulation of the phosphorylation of elongation factor 2 by MEK-dependent signalling in adult rat cardiomyocytes

The Gq-coupled agonists phenylephrine and endothelin-1 each activate protein synthesis in cardiomyocytes as part of the programme that leads to cardiac hypertrophy. Here we show that they each induce the dephosphorylation of elongation factor (eEF) 2, a protein that in its dephosphorylated state mediates the translocation step of elongation. The ability of both agonists to induce dephosphorylation of eEF2 requires signalling via the mTOR and MEK/Erk signalling pathways, but is independent of phosphoinositide 3-kinase. Expression of an activated form of MEK leads to dephosphorylation of eEF2, in an mTOR independent manner, indicating that signalling via MEK/Erk suffices to cause dephosphorylation of eEF2.     

Immunocytochemical analysis of the regeneration of myofibrils in long-term cultures of adult cardiomyocytes of the rat

Dissociated adult rat ventricular cardiomyocytes obtained from hearts by retrograde perfusion with collagenase were investigated in long-term cultures. Myofibril regeneration, isoprotein transition of alpha- and beta-myosin heavy chain (MHC), and M-band localization of M-creatine kinase in the reconstituting heart cells were studied. Myofibril formation was demonstrated by the use of antibodies against either cardiac C-protein or myomesin as early differentiation markers. Four days after plating, small myofibrils could be identified in attached cells in a perinuclear fashion; later in culture the cells displayed various shapes and myofibril distribution. Frequently a patchy distribution of myofibrils within the extending peripheral processes could be observed. Colocalization of sarcomeres and phalloidin-stained F-actin filament bundles was demonstrated by double fluorescence staining and by the use of high intensifying video microscopy and computerized image processing. The immunofluorescence distribution of alpha- and beta-MHC isoproteins in newly isolated and cultured cardiomyocytes changed from 100% alpha-MHC and 70% beta-MHC in rod-shaped cells to about 100% beta-MHC and 70% alpha-MHC in spread out cultured cells. This shift was corroborated by a relative gradual decline in alpha-MHC at the expense of increasing amounts of beta-MHC with time in culture as assessed by sodium dodecyl sulfate gel electrophoresis of total cell homogenates. In addition, whereas rod-shaped newly isolated cardiomyocytes showed a clear M-band association of M-creatine kinase as found in adult heart tissue, adult cultivated spread out cells did not show a cross-striated pattern after incubation with antibody. Taken together, these observations suggest that adult cardiomyocytes not only undergo extensive morphological transitions in long-term cultures, but also generate new myofibrillar structures lacking M-creatine kinase and containing the beta-MHC, thus fitting the characteristics of fetal myofibrils. These results indicate a change from the adult terminally differentiated to a less differentiated state of the cardiac cells in culture.     

Cardioprotective action of CRF peptide urocortin against simulated ischemia in adult rat cardiomyocytes

The major objective of this study was to determine whether urocortin, a member of the corticotrophin-releasing factor (CRF) family, protects adult rat cardiomyocytes from ischemia that has been simulated by glucose deprivation and acidosis. When it was present during simulated ischemia, urocortin (0.1 microM) markedly attenuated the cellular injury, which was assessed by increases in creatine kinase and lactate dehydrogenase levels. This effect was comparable with that observed with adenosine (10 microM). The cardioprotective effect of urocortin was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate, an inhibitor of ATP-sensitive K(+) channels. Cardiomyocytes were also protected from injury by pretreatment with urocortin, either by incubation for 5 min with a subsequent 10-min recovery or incubation for 20 min with a 20-h recovery before simulated ischemia. Similar cardioprotective effects were observed with ischemic preconditioning protocols during both immediate and delayed phases. In conclusion, in adult cardiomyocytes, urocortin has immediate and delayed cardioprotective actions that mimic ischemic preconditioning. These actions are mediated via protein kinase C and ATP-sensitive K(+) channels.     

Effects of aging on the lateral transmission of force in rat skeletal muscle

The age-related reduction in muscle force cannot be fully explained by the loss of muscle fiber mass or degeneration of myofibers. Our previous study showed that changes in lateral transmission of force could affect the total force transmitted to the tendon. The extracellular matrix (ECM) of skeletal muscle plays an important role in lateral transmission of force. The objective of this study was to define the effects of aging on lateral transmission of force in skeletal muscles, and explore possible underlying mechanisms. In vitro contractile tests were performed on extensor digitorum longus (EDL) muscle of young and old rats with series of tenotomy and myotomy. We concluded that lateral transmission of force was impaired in the old rats, and this deficit could be partly due to increased thickness of the ECM induced by aging.     

Plasma fibronectin-binding glycosaminoglycans in the substratum adhesion sites of neural tumor cells

The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.     

成年大鼠心肌细胞分离培养及兴奋-收缩耦联表征

目的：比较两种细胞分离液分离成年大鼠心肌细胞,进一步表征成年大鼠心室肌细胞兴奋-收缩耦联。方法 Langendorff装置进行主动脉逆流灌流,分别用两种细胞分离液分离成年大鼠心肌细胞,无血清培养并进行腺病毒感染。显微镜下观察单个心肌细胞的形态学特点,荧光显微镜下检测病毒感染。采用IonOptix仪器检测心肌细胞肌节收缩-舒张指标以及心肌细胞钙离子摄入-排出指标。结果两种分离液均可获得70％横纹清晰的长杆状心肌细胞,培养可存活7 d以上。腺病毒感染48 h,绿色荧光蛋白持续表达7 d以上。分离液一获得的心肌细胞不能很好地随电场刺激产生收缩,分离液二获得的细胞可用于检测兴奋-收缩耦联特性,心肌细胞肌节缩短分数为11.61％±2.15％,舒张时间为（0.177±0.031） s,钙瞬变幅度为30.79％±9.74％,钙瞬变衰减时间为（0.300±0.074） s。结论两种分离液均可用于分离和培养成年大鼠心肌细胞,并用于腺病毒转染等长时程研究。分离液二更适用于检测成年大鼠心肌细胞的兴奋-收缩耦联特性。     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.