Fluctuations of Contraction Rhythm During Simulated Ischemia/Reperfusion in Cultured Cardiac Myocytes from Neonatal Rats

Cardiac ischemia results in a rapid decrease of intracellular pH and in the rise of intracellular Ca 2+ , changes that have been shown to reduce intercellular communication via gap junctions (GJ) between cardiac myocytes. Ischemia also results in electrical instability probably caused by the reduced GJ permeability contributing to an increased vulnerability to arrhythmias. This study aims at elucidating whether the fluctuations of contraction rhythm of spontaneously beating cardiac myocytes in culture changes during simulated ischemia/reperfusion. The coefficient of variation (CV) of contraction intervals, reflecting the fluctuation of contraction rhythm, increased significantly during simulated ischemia/reperfusion. However, the contraction rhythm of the cardiac myocytes in an aggregate remained synchronized during simulated ischemia/reperfusion. In contrast, pharmacological blockade of GJ with 12-doxyl stearic acid, a blocker of GJ permeability, resulted in the de-synchronization of contraction rhythm and in an increase in the CV of contraction intervals in normoxic conditions. The present findings lead to the suggestion that GJ remained open during simulated ischemia/reperfusion, and that a mechanism other than electrical uncoupling between myocytes contributed to the observed increase in the fluctuation of beating rhythm during ischemia.     

培养新生大鼠心肌细胞的电信号传导：多电极记录研究

利用多电极阵列同步记录技术对培养的新生大鼠单层心肌细胞的电活动进行胞外记录,观察心肌细胞在自发搏动和电刺激情况下信号在细胞间的传导模式。通过对记录信号的处理和分析,能获得诸如起搏细胞的数量和位置、动作电位的传导速度和途径以及不同起搏细胞间的相互影响等信息。研究还发现,心肌细胞阈下刺激会影响细胞的搏动和信号传导。     

Reversible Inhibition of Gap Junctional Intercellular Communication, Synchronous Contraction, and Synchronism of Intracellular Ca Fluctuation in Cultured Neonatal Rat Cardiac Myocytes by Heptanol

We analyzed by Fotonic Sensor, a fiber-optic displacement measurement instrument, the effects of heptanol on synchronized contraction of primary neonatal rat cardiac myocytes cultured at confluent density. We also examined the effect of heptanol on the changes in gap junctional intercellular communication by using the microinjection dye transfer method, and on intercellular Ca²⁺ fluctuation by confocal laser scanning microscopy of myocytes loaded with the fluorescent Ca²⁺ indicator fluo 3. In addition, we studied expression, phosphorylation, and localization of the major cardiac gap junction protein connexin 43 (Cx43) using immunofluorescence and Western blotting. At Day 6 of culture, numerous myocytes exhibited spontaneous, synchronous contractions, excellent dye coupling, and synchronized intracellular Ca²⁺ fluctuations. We treated the cells with 1.5, 2.0, 2.5, and 3.0 mmol/liter heptanol. With 1.5 mmol/liter heptanol, we could not observe significant effects on spontaneous contraction of myocytes. At 3.0 mmol/liter, the highest concentration used in the current experiment, heptanol inhibited synchronous contractions and even after washing out of heptanol, synchronous contraction was not rapidly recovered. On the other hand, at the intermediate concentrations of 2.0 and 2.5 mmol/liter, heptanol reversely inhibited synchronized contraction, gap junctional intercellular communication, and synchronization of intracellular Ca²⁺ fluctuations in the myocytes without preventing contraction and changes of intracellular Ca²⁺ in individual cells. Brief exposure (5-20 min) to heptanol (2.0 mmol/liter) did not cause detectable changes in the expression, phosphorylation, or localization of Cx43, despite strong inhibition of gap junctional intercellular communication. These results suggest that gap junctional intercellular communication plays an important role in synchronous intracellular Ca²⁺ fluctuations, which facilitate synchronized contraction of cardiac myocytes.     

Fluctuations in the Concentration of Extracellular ATP Synchronized with Intracellular Ca2+ Oscillatory Rhythm in Cultured Cardiac Myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The intracellular concentration of free Ca²⁺ also changes rhythmically in association with the rhythmic contraction of myocytes (Ca²⁺ oscillation). Both the contraction and Ca²⁺ oscillatory rhythms are synchronized among myocytes, and intercellular communication via gap junctions has been considered primarily responsible for the synchronization. However, a recent study has demonstrated that intercellular communication via extracellular ATP‐purinoceptor signaling is also involved in the intercellular synchronization of intracellular Ca²⁺ oscillation. In this study, we aim to elucidate whether the concentration of extracellular ATP changes cyclically and contributes to the intercellular synchronization of Ca²⁺ oscillation among myocytes. In almost all the cultured cardiac myocytes at four days in vitro (4 DIV), intracellular Ca²⁺ oscillations were synchronized with each other. The simultaneous measurement of the concentration of extracellular ATP and intracellular Ca²⁺ revealed the extracellular concentration of ATP actually oscillated concurrently with the intracellular Ca²⁺ oscillation. In addition, power spectrum and cross‐correlation analyses suggested that the treatment of cultured cardiac myocytes with suramin, a blocker of P2 purinoceptors, resulted in the asynchronization of Ca²⁺ oscillatory rhythms among cardiac myocytes. Treatment with suramin also resulted in a significant decrease in the amplitudes of the cyclic changes in both intracellular Ca²⁺ and extracellular ATP. Taken together, the present study demonstrated the possibility that the concentration of extracellular ATP changes cyclically in association with intracellular Ca²⁺, contributing to the intercellular synchronization of Ca²⁺ oscillation among cultured cardiac myocytes.     

Rhythmic Fluctuations in the Concentration of Intracellular Mg2+ in Association with Spontaneous Rhythmic Contraction in Cultured Cardiac Myocytes

Magnesium ions (Mg²⁺) play a fundamental role in cellular function, but the cellular dynamic changes of intracellular Mg²⁺ remain poorly delineated. The present study aims to clarify whether the concentration of intracellular Mg²⁺ possibly changes cyclically in association with rhythmic contraction and intracellular Ca²⁺ oscillation in cultured cardiac myocytes from neonatal rats. To do this, we performed a noise analysis of fluctuations in the concentration of intracellular Mg²⁺ in cardiac myocytes. The concentration was estimated by loading cells with either Mg‐fluo4/AM or KMG‐20/AM. Results revealed that the intensity of Mg‐fluo‐4 or KMG‐20 fluorescence fluctuated cyclically in association with the rhythmic contraction of cardiac myocytes. In addition, the simultaneous measurement of Fura2 and Mg‐fluo‐4 fluorescence revealed phase differences between the dynamics of the two signals, suggesting that the cyclic changes in the Mg‐fluo‐4 or KMG‐20 fluorescent intensity actually reflected the changes in intracellular Mg²⁺. The complete termination of spontaneous rhythmic contractions did not abolish Mg²⁺ oscillations, suggesting that the rhythmic fluctuations in intracellular Mg²⁺ did not result from mechanical movements. We suggest that the concentration of intracellular Mg²⁺ changes cyclically in association with spontaneous, cyclic changes in the concentration of intracellular Ca²⁺ of cardiac myocytes. A noise analysis of the fluctuation of subtle changes in fluorescence intensity could contribute to the elucidation of novel functional roles of Mg²⁺ in cells.     

The Expression, Phosphorylation, and Localization of Connexin 43 and Gap-Junctional Intercellular Communication during the Establishment of a Synchronized Contraction of Cultured Neonatal Rat Cardiac Myocytes

We analyzed the expression, phosphorylation, and localization of the major cardiac gap-junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluent monolayers of primary cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Monitoring of the beating rate and synchronization by Fotonic Sensor showed that at Day 1 of culture cardiac myocytes contracted spontaneously but irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, the confluent cells exhibited synchronous contraction with a relatively constant rate (125 ± 20 beats/min). Cardiac myocytes expressed a large amount of Cx43 mRNA even at Day 1 and maintained the expression until at least Day 7. Immunofluorescence of Cx43 showed that the localization of Cx43-positive spots was mostly restricted to cell-cell contacts between myocytes and that few Cx43-positive spots were present between myocytes and fibroblasts or between fibroblasts. The amount of Cx43 protein, the proportion of phosphorylated forms to the nonphosphorylated one, and the number and total area of Cx43-positive spots increased with culture time. Gap-junctional intercellular communication measured by dye transfer assay was also increased with culture time and correlated well with the number and total area of Cx43-positive spots. Our systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercellular communication plays a major role in the reestablishment of synchronous beating of cultured neonatal rat cardiac myocytes.     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.     

Muscle Differentiation in Cultured Pineal Body Cells of Neonatal Rats

Dissociated cells of pineal bodies of new-born rats were cultured to see what cell types would be differentiated during culture in vitro for about 4 weeks. In early stages of culture, about 10 days after inoculation, flattened cells with piliform processes, small round cells and small bubbling cells were distinguishable in the cultures. After about 2 weeks, neuronal cells with axon-like processes and multinuclear muscle-like cells were differentiated. On further culture, the latter cells differentiated into mature striated myotubes. The developmental origin of myotube formed in cell cultures of pineal body is discussed.     

Differential Effects of Hypoxic and Hyperoxic Stress-Induced Hypertrophy in Cultured Chick Fetal Cardiac Myocytes

The adult heart responds to contraction demands by hypertrophy, or enlargement, of cardiac myocytes. Adaptive hypertrophy can occur in response to hyperoxic conditions such as exercise, while pathological factors that result in hypoxia ultimately result in heart failure. The difference in the outcomes produced by pathologically versus physiologically induced hypertrophy suggests that the cellular signaling pathways or conditions of myocytes may be different at the cellular level. The structural and functional changes in myocytes resulting from hyperoxia (simulated using hydrogen peroxide) and hypoxia (using oxygen deprivation) were tested on fetal chick cardiac myocytes grown in vitro. Structural changes were measured using immunostaining for α-sarcomeric actin or MyoD, while functional changes were assessed using immunostaining for calcium/calmodulin-dependent kinase (CaMKII) and by measuring intracellular calcium fluxes using live cell fluorescence imaging. Both hypoxic and hyperoxic stress resulted in an upregulation of actin and MyoD expression. Similarly, voltage-gated channels governing myocyte depolarization and the regulation of CaMK were unchanged by hyperoxic or hypoxic conditions. However, the dynamic features of calcium fluxes elicited by caffeine or epinephrine were different in cells subjected to hypoxia versus hyperoxia, suggesting that these different conditions differentially affect components of ligand-activated signaling pathways that regulate calcium. Our results suggest that changes in signaling pathways, rather than structural organization, may mediate the different outcomes associated with hyperoxia-induced versus hypoxia-induced hypertrophy, and these changes are likely initiated at the cellular level.     

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state. Received: 10 April 1995/Revised: 27 October 1995     

Eicosapentaenoic Acid Ameliorates Cardiac Fibrosis and Tissue Inflammation in Spontaneously Hypertensive Rats

Hypertension affects 1 in 3 adults in the United States and leads to left ventricular (LV) concentric hypertrophy, interstitial fibrosis, and increased stiffness. The treatment of cardiac fibrosis remains challenging and empiric. Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that is highly effective in reducing cardiovascular events in patients and cardiac fibrosis and hypertrophy in animals when administered before pressure overload by promoting the increase of anti-inflammatory M1 macrophages. In this study, we investigated whether EPA mitigates the exacerbation of cardiac remodeling and fibrosis induced by established hypertension, a situation that closely recapitulates a clinical scenario. Twelve-week-old spontaneously hypertensive rats were randomized to eat an EPA-enriched or control diet for 20 weeks. We report that rats eating the EPA-enriched diet exhibited a reduction of interstitial cardiac fibrosis and ameliorated LV diastolic dysfunction despite the continuous increase in blood pressure. However, we found that EPA did not have an impact on cardiac hypertrophy. Interestingly, the EPA diet increased mRNA expression of M2 macrophage marker Mrc1 and interleukin-10 in cardiac tissue. These findings indicated that the antifibrotic effects of EPA are mediated in part by phenotypic polarization of macrophages toward anti-inflammatory M2 macrophages and increases of the anti-inflammatory cytokine, interleukin-10. In summary, EPA prevents the exacerbation of cardiac fibrosis and LV diastolic dysfunction during sustained pressure overload. EPA could represent a novel treatment strategy for hypertensive cardiomyopathy.     

细胞周期素D在培养的新生大鼠心肌细胞中的表达

目的探索一种快速分离和培养新生大鼠心肌细胞的方法,研究心肌细胞中细胞周期素D表达的变化规律.方法心肌细胞分离、纯化及培养后用倒置显微镜观察其形态变化,免疫细胞化学染色及图像分析方法检测培养第1～21天新生大鼠心室肌细胞周期素D的表达.结果心肌细胞核内细胞周期素D表达量逐渐下降,第2天与第1天比较,差异无显著性(P>0.05),从第3天开始差异有显著性(P<0.05,P<0.01).结论新生大鼠心肌细胞出生后早期体外培养的前3天有一定增殖能力,但以后呈下降趋势.     

Protective Role of Acidic pH-Activated Chloride Channel in Severe Acidosis-Induced Contraction from the Aorta of Spontaneously Hypertensive Rats

Severe acidic pH-activated chloride channel (I_Cl,acid) has been found in various mammalian cells. In the present study, we investigate whether this channel participates in reactions of the thoracic aorta to severe acidosis and whether it plays a role in hypertension. We measured isometric contraction in thoracic aorta rings from spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. Severe acidosis induced contractions of both endothelium-intact and -denuded thoracic aorta rings. In Wistar rats, contractions did not differ at pH 6.4, 5.4 and 4.4. However, in SHRs, contractions were higher at pH 5.4 or 4.4 than pH 6.4, with no difference between contractions at pH 5.4 and 4.4. Nifedipine, I_Cl,acid blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS) inhibited severe acidosis-induced contraction of aortas at different pH levels. When blocking I_Cl,acid, the remnant contraction was greater at pH 4.4 than pH 5.4 and 6.4 for both SHRs and Wistar rats. With nifedipine, the remnant contraction was greatly reduced at pH 4.4 as compared with at pH 6.4 and 5.4. With NPPB or DIDS, the ratio of remnant contractions at pH 4.4 and 5.4 (R_4.4/5.4) was lower for SHRs than Wistar rats (all <1). However, with nifedipine, the R_4.4/5.4 was higher for SHRs than Wistar rats (both >1). Furthermore, patch clamp recordings of I_Cl,acid and intracellular Ca²⁺ measurements in smooth muscle cells confirmed these findings. I_Cl,acid may protect arteries against excess vasoconstriction under extremely acidic extracellular conditions. This protective effect may be decreased in hypertension.     

Mechanotransduction in Stretch-Induced Hypertrophy of Cardiac Myocytes

Abstract

Mechanical loading of cardiac muscles causes rapid activation of a number of immediate-early (IE) genes and hypertrophy. However, little is known as to how muscle cells sense mechanical load and regulate gene expression. We examined roles of several putative mechanotransducers in stretch-induced hypertrophy of cardiac myocytes grown on a deformable silicone sheet. Using the patch-clamp technique, we found a single class of stretch-activated cation channels which was completely and reversibly blocked by gadolinium. The inhibition of this channel by gadolinium did not affect either stretch-induced expression of the IE genes or hypertrophy. Neither disruption of microtubules with colchicine nor that of actin microfilaments by cytochalasin D prevented the stretch-induced IE gene expression. Arresting contractile activity by tetrodotoxin did not affect the stretch-induced IE gene expression or hypertrophy. These results suggest that stretch-activated cation channels, microtubules, microfilaments, and contractile activity are not the mechanotransducers. Preliminary results suggest that cell stretch may cause a release of a growth factor(s), which in turn initiates a cascade of hypertrophic response of cardiac myocytes.     

Effect of Daunorubicin on Subcellular Pools of Glutathione in Cultured Heart Cells from Neonatal Rats

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase.

Control heart cells were found to contain 12.2 ± 1.8 nmolesGSH/IO⁶ cells. Daunorubicin causedarapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH.

By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 89 ± 1.5nmoles, 10 cells and 3.3 ± 0.6 nmoles/10⁶ cells. respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and. after a short lag period. a release of lactate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH.

No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase. whereas dithiothreitol offered partial protection.

The results provide support for an oxidative mechanism in which the decrease in the cytosolic pool of GSH may be the causative factor of daunorubicin-induced toxicity. This decrease in GSH may affect the cytosolic NADPH and various redox groups on proteins, thereby altering the permeability of the plasma membrane and finally causing cell damage.     

Pharmacological Inhibition of AMP-Deaminase in Rat Cardiac Myocytes

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC ₅₀ = 0.5 μ M. AMPDI was much less effective with isolated cardiomyocytes (IC ₅₀ = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.     

自发性高血压大鼠血管平滑肌及心肌内成纤维细胞生长特性的研究

目的　探讨自发性高血压大鼠(spontaneously hypertensive rats, SHR)主动脉平滑肌细胞(aorta smooth muscle cell, ASMC)与心肌内成纤维细胞(cardiac fibroblast, CFB)在体外培养生长的特性。方法　16周龄自发性高血压大鼠(SHR)和正常大鼠(WKY)测血压后处死,取心脏与胸主动脉,组织块法分别培养CFB与ASMC)。分别观察(1)CFB     

The Inhibition of N-Glycosylation of Glycoprotein 130 Molecule Abolishes STAT3 Activation by IL-6 Family Cytokines in Cultured Cardiac Myocytes

Interleukin-6 (IL-6) family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130), a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK)1/2 through janus kinases (JAKs); however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm), an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF)-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs) and protein-tyrosine phosphatase 1B (PTP1B), which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R) could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.