Chronic sympathetic activation promotes downregulation of β-adrenoceptor-mediated effects in the guinea pig heart independently of structural remodeling and systolic dysfunction

It is uncertain if downregulation of β-adrenoceptor signaling pathway is promoted by an enhanced adrenergic tone at an early stage of cardiac disease, or it develops secondary to detrimental local myocardial changes in advanced heart failure. We examined the integrity of β-adrenoceptor signaling pathway upon chronic infusion of isoproterenol, a β-adrenoceptor agonist, at a dose producing no structural left ventricular (LV) remodeling and systolic dysfunction. Subcutaneous isoproterenol infusion (400 μg kg⁻¹ h⁻¹ over 16 days) to guinea pigs using osmotic minipumps produced no change in cardiac weights, LV internal dimensions, myocyte cross-sectional area, extent of interstitial fibrosis, and basal contractile function. Isolated, perfused heart preparations from isoproterenol-treated guinea pigs exhibited attenuated responsiveness to acute β-adrenoceptor stimulation, as evidenced by reduced LV developed pressure increase, less shortening of LV epicardial monophasic action potential and effective refractory period, and less myocardial cyclic adenosine monophosphate elevation, in response to isoproterenol exposure, when compared to saline-treated controls. Pharmacological responses to forskolin, an activator of the adenylate cyclase catalytic subunit, were well preserved in isoproterenol-treated hearts. Downregulation of β-adrenoceptor-mediated effects upon chronic isoproterenol infusion was associated with markedly reduced stimulatory G-protein α-subunit (G_sα) myocardial expression levels. No change in expression levels of β-adrenoceptors, G-protein-coupled receptor kinase 2, inhibitory G-protein α-subunit (G_iα2), and Ca_v1.2 and K_v7.1 ion channels was determined in isoproterenol-treated hearts. We therefore conclude that sustained adrenergic overstimulation may promote downregulation of myocardial β-adrenoceptor-mediated effects independently of structural LV remodeling and systolic failure, an effect attributed to β-adrenoceptor uncoupling from adenylate cyclase due to reduced G_sα-protein expression.     

Role of adrenoreceptors of Ehrlich's ascitic carcinoma in the regulation of proliferative processes by adrenergic ligands

Two categories of specific binding sites for the β-adrenergic ligand³H-dihydroalprenolol have been revealed on the cells of Ehrlich's ascitic carcinoma. The synthesis of DNA in the tumor cell culture possessed different sensitivities to α- and β-adrenergic preparations. Propranolol, a β-adrenoblocker, suppressed DNA synthesis most intensively in concentrations approaching the therapeutic ones. It is hypothesized that β-adrenoreceptors participate predominantly in the regulation of proliferative processes in Ehrlich's ascitic carcinoma by adrenergic ligands. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N^no 11, pp. 525–528, November, 1995     

激活TGR5通过CaN/NFAT3途径减轻高糖诱导的心肌细胞肥大

目的:观察激活G蛋白偶联胆汁酸受体1(GPBAR1,又称TGR5)对高糖诱导的小鼠心肌肥大的影响,并探讨钙调神经磷酸酶(CaN)/活化T细胞核因子3(NFAT3)信号途径在其中的作用。方法:原代培养小鼠心肌细胞,采用图像分析系统测定细胞表面积,BCA法测定细胞蛋白含量,通过RT-PCR及Western blot方法检测TGR5、CaN及NFAT3的mRNA及蛋白表达变化。结果:成功培养小鼠心肌细胞。高糖明显诱导心肌细胞表面积及细胞蛋白含量的增加(P0.05)同时CaN及NFAT3的表达也增加(P0.05)。激活TGR5或给予CaN抑制剂环孢素A均能抑制高糖引起的心肌细胞肥大及NFAT3表达增加(P0.05)。给予TGR5干扰慢病毒可阻断TGR5对心肌细胞肥大的上述改善作用(P0.05)。结论:激活TGR5能减轻高糖诱导的心肌细胞肥大,其机制可能与抑制CaN/NFAT3信号通路有关。     

Increased Ca2+ sensitivity and protein expression of SERCA 2a in situations of chronic β3-adrenoceptor deficiency

This study investigated the influence of chronic β₃-adrenoceptor deficiency on myocardial function. Therefore, we investigated Ca²⁺-regulatory proteins, SERCA 2a activity, and myofibrillar and mitochondrial function in hearts of wild-type (WT, n=7) and β₃-adrenoceptor knockout mice (β₃-KNO, n=7). Morphometric heart analysis showed no difference between WT and β₃-KNO. No alterations were observed for the protein expression of the ryanodine receptor or phospholamban. However, in β₃-KNO mice, protein expression of SERCA 2a and phospholamban phosphorylation were significantly increased. These changes were accompanied by an increased SERCA 2a activity in β₃-KNO. Alterations in phospholamban phosphorylation were independent of alterations in β₁/β₂-adrenoceptor distribution and protein expression of G proteins in β₃-KNO. Measurement of myofibrillar Ca²⁺ sensitivity showed no difference in the Ca²⁺/force relation for WT and β₃-KNO. The same seems to hold true for mitochondrial function since the protein expressions of cytochrome c, uncoupling protein 3 and cytochrome c oxidase subunit IV were similar in WT and β₃-KNO. The conclusion is that depression of β₃-adrenergic stimulation may modulate the protein expression of SERCA 2a and phospholamban phosphorylation, thereby improving sarcoplasmic reticulum Ca²⁺ uptake. Thus, β₃-adrenergic depression may be a therapeutic aim in situations of impaired SERCA 2a activity, e.g. for the treatment of heart failure.     

Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis

A noninvasive method for the characterization of cardiomyocyte contractile behavior is presented. Light microscopic video images of cardiomyocytes were captured with a high-speed camera, and motion vectors (which have a velocity dimension) were calculated with a high spatiotemporal resolution using a block-matching algorithm. This method could extract contraction and relaxation motions of cardiomyocytes separately and evaluate characteristics such as the beating rate, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and wave propagation of impulses. Simultaneous phase-contrast imaging and calcium (Ca2+) fluorescence measurements confirmed that the timing of the maximum shortening velocity of cardiomyocytes correlated well with intracellular Ca2+ transients. Based on our analysis, gap junction inhibitors, 1-heptanol (2?mM) or 18-β-glycyrrhetinic acid (30?μM), resulted in clear changes in beating cooperativity and the propagation pattern of impulses in the cardiomyocyte monolayer. Additionally, the time dependence of the motion vector length indicated a prolonged relaxation process in the presence of potassium (K+) channel blockers, dl-sotalol (1?μM), E-4031 (100?nM), or terfenadine (100?nM), reflecting the prolonged QT (Q wave and T wave) interval of cardiomyocytes. Effects of autonomic agents (acetylcholine or epinephrine [EPI]) or EPI and propranolol on cardiomyocytes were clearly detected by the alterations of beating rate and the motion vector length in contraction and relaxation processes. This method was noninvasive and could sensitively evaluate the contractile behavior of cardiomyocytes; therefore, it may be used to study and/or monitor cardiomyocyte tissue during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes.     

Unchanged sarcoplasmic reticulum Ca2+-ATPase activity, reduced Ca2+ sensitivity, and negative force-frequency relationship in transgenic rats overexpressing the mouse renin gene

Transgenic rats overexpressing the mouse Ren-2 gene [TG(mREN2)27 rats, TGR] were used to characterize alterations in force generation and relaxation following cardiac hypertrophy. Age-matched Sprague-Dawley rats were used as the control group. The β-adrenoceptor dependent increase in force of contraction was reduced in the transgenic animals but not the Ca²⁺-dependent increase in force generation. Additionally, force of contraction decreased after increasing stimulation frequencies (up to 7 Hz), but the frequency-dependent decrease in force of contraction was significantly more pronounced in the transgenic group. The Ca²⁺ sensitivity in chemically skinned fiber preparations of TGR was reduced than that in Sprague-Dawley rats while maximum effectiveness was the same. Unexpectedly, the sarcoplasmic reticulum Ca²⁺-ATPase activity measured in crude membrane preparations from TGR did not differ from that in Sprague-Dawley rats; however, the activity of the Na⁺/K⁺-ATPase was less while the Na⁺/Ca²⁺-exchanger activity was significantly greater. In the same preparations the protein expression of SERCA2 was reduced in TGR while expression of phospholamban and calsequestrin remained the same. Thus in the model of cardiac hypertrophy harboring the mouse Ren-2 gene the hypothesized correlation between SERCA2 function and force-frequency relationship was not observed. Possible reasons for the more negative force-frequency relationship in TGR included changes at the level of the myofilaments and altered intracellular Na⁺ homeostasis which may result from the reciprocal changes in the Na⁺/K⁺-ATPase and the Na⁺/Ca²⁺-exchanger activity. Received: 15 September 1997 / Accepted: 7 April 1998     

Rate‐dependent electrical,contractile and restitution properties of isolated left ventricular myocytes in guinea‐pig hypertrophy

Left ventricular hypertrophy predisposes to sudden cardiac death (SCD) and studies of human SCD suggest that the antecedent heart rate (HR) is usually <100 beats min^–1. This is surprising in view of the known association between adrenergic receptor stimulation and SCD which by itself would suggest that it is more likely to occur from high rather than low HR. We therefore hypothesized that there may be electrical or mechanical abnormalities present in myocytes isolated from animals with left ventricular hypertrophy that predispose to SCD at low stimulation frequencies but which may not be present at high HR. Mild left ventricular hypertrophy was induced in guinea‐pigs by infra‐renal aortic banding. Electrical and mechanical properties of isolated myocytes were studied at different stimulation frequencies between 0.1 and 3 Hz. Action potential duration (APD) is prolonged in hypertrophy at stimulation frequencies <1 Hz but not at faster rates. Contraction size, time‐to‐peak contraction (TTPC) and half‐relaxation time are greatly enhanced in hypertrophy at all frequencies between 0.1 and 3 Hz. Electrical (50.3 ± 5.2 ms in hypertrophy and 78.4 ± 12.1 ms in control, P < 0.03) and mechanical (205 ± 16 ms for hypertrophy and 266 ± 24 ms for control cells, P < 0.03) restitution time constants are quicker in hypertrophy. The finding of APD prolongation at low but not at high frequencies is consistent with the finding that SCD arises from low and not high HR. This data supports the role of abnormal repolarization in SCD.     

Adrenergic Regulation of Erythropoiesis during Cytostatic-Induced Myelosuppressions

The role of central adrenergic structures in the regulation of the erythroid hematopoietic stem was studied during administration of cyclophosphamide and 5-fluorouracil. The central nervous system contributed to suppression of erythropoiesis during cytostatic treatment. The suppressive effect of brain adrenergic structures on the erythron after treatment with cyclophosphamide and 5-fluorouracil was related to dysfunction of adherent cells in the hemopoiesis-inducing microenvironment (formation of hemopoietic islets and secretion of erythropoietic activity) and production of growth factors by myelokaryocytes, respectively. Brain norepinephrine had an inhibitory effect on proliferative activity and differentiation of erythroid precursors that were associated with the erythropoietin and peripheral α-adrenergic structures. However, stimulation of β-adrenergic structures was followed by an increase in the rate of erythroid cell maturation. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 10, pp. 385–390, October, 2008     

Temporal changes in myocardial adrenergic regulation with the progression to pump dysfunction after chronic β-adrenoreceptor activation in rats

We evaluated the relationship between myocardial norepinephrine release or inotropic responsiveness to adrenergic stimulation and intrinsic myocardial function after the progression to pump dysfunction induced by chronic β-adrenoreceptor activation (isoproterenol [ISO], 0.1 mg/kg/day for 1 month or 6 months) in rats. Left ventricular (LV) systolic chamber dysfunction occurred after 6 months, but not after 1 month of β-adrenoreceptor activation, as evidenced by reduced LV endocardial fractional shortening determined by echocardiography and a decrease in the slope of the LV systolic pressure–volume relations assessed in isolated, perfused heart preparations. A reduced pump function at 6 months of ISO administration was associated with chamber dilatation, while LV midwall fractional shortening (echocardiography) and the slope of the LV systolic stress–strain relations (isolated heart preparations), indices of intrinsic myocardial function, were unchanged. After 1 month of ISO administration, reduced β₁- and β₂-adrenoreceptor-mediated and sustained α-adrenoreceptor-mediated inotropic responses were noted. Nevertheless, increased inotropic potency of β-adrenoreceptor agonists and upregulation of α-adrenoreceptor-mediated contractile responses were noted after 6 months of ISO administration. Increased adrenergic–inotropic responsiveness after 6 months of ISO administration was associated with depleted LV norepinephrine stores, as evidenced by reduced desipramine-stimulated norepinephrine concentrations in the coronary effluent. In conclusion, in the progression from compensated cardiac hypertrophy to pump dysfunction after chronic sympathetic activation, a preserved intrinsic myocardial contractility is accounted for by paradoxical upregulation of adrenergic-mediated contractile responses.     

Spinophilin is required for normal morphology, Ca2+ homeostasis and contraction but dispensable for ��-adrenergic stimulation of adult cardiomyocytes

Spinophilin (SPN) is a ubiquitously expressed scaffolding protein that interacts through several binding modules with a variety of target proteins. Thus, SPN bundles F-actin, targets protein phosphatase 1 to the ryanodine receptor, and targets regulators of G-protein signaling to G-protein coupled receptors in cardiomyocytes. In this work we studied the role of SPN on cardiomyocyte morphology, function, and β-adrenergic responsiveness using a homozygous SPN knock-out mouse model (SPN-/-). We show that spinophilin deficiency significantly (1) reduced cardiomyocyte length, (2) increases both Ca(2+) amplitude and maximal rate of Ca(2+) rise during systole, and (3) decreased shortening amplitude and maximal rate of shortening, while (4) β-adrenergic stimulation remained intact. Our data suggest that spinophilin is an upstream regulator required for normal growth and excitation-contraction coupling, but is dispensable for β-adrenergic stimulation of adult cardiomyocytes.     

Adrenergic signalling between rat taste receptor cells

In taste buds, synaptic transmission is traditionally thought to occur from taste receptor cells to the afferent nerve. This communication reports the novel observation that taste receptor cells respond to adrenergic stimulation. Noradrenaline application inhibited outward potassium currents in a dose-dependent manner. This inhibition was mimicked by the β agonist isoproterenol and blocked by the β antagonist propranolol. The α agonists clonidine and phenylephrine both inhibited the potassium currents and elevated intracellular calcium levels. Inwardly rectifying potassium currents were unaffected by adrenergic stimulation. Experiments using the RT-PCR technique demonstrate that lingual epithelium expresses multiple α (α1a, α1b, α1c, α1d, α2a, α2b, α2c) and β (β1, β2) subtypes of adrenergic receptors, and immunocytochemistry localized noradrenaline to a subset of taste receptor cells. Collectively, these data imply strongly that adrenergic transmission within the taste bud may play a paracrine role in taste physiology.     

Progressive arteriolar vasoconstriction and fatigue during tetanic contractions of rat skeletal muscle are inhibited by α-receptor blockade

Voluntary muscle contractions activate sympathetic efferent pathways. Using a fatiguing electrical stimulation protocol designed specifically to enhance sympathetically-mediated vasoconstrictor tone, we explored the temporal profile and mechanistic bases of the evoked vasoconstrictor response and its role in muscle fatigue. Spinotrapezius muscles of Wistar rats were exteriorized and stimulated tetanically (100 Hz, 6–8 V, stimulus duration 700 ms) every 3 s for 2.5 min. The extent and time course of diameter changes in arterioles (1A and 2A) and venules (1V and 2V) were determined after each of 10 discrete sets of muscle stimulation at 5-min intervals. At first, to compare the effect of stimulation parameters in this preparation, stimulations were performed with rectangular pulses of either 0.2- or 4-ms pulse duration. As expected the 0.2-ms pulse stimulation did not affect arteriolar diameter or muscle fatigability. In contrast, during and following 4-ms pulse stimulations, there was a surprising arteriolar vasoconstriction rather than the expected vasodilation. Arteriolar (but not venular) vasoconstriction (reduced arteriolar diameter by 38.6 ± 2.6% in the 10th set) increased progressively with muscle fatigue (to 29 ± 12% of initial tension in the 10th set) for the 4-ms pulse condition. Superfusion with the selective α1-adrenergic receptor antagonist prazosin (1 μM) and/or α2-adrenergic receptor antagonist rauwolscine (10 μM) abolished both the arteriolar vasoconstriction and significantly reduced fatigue (i.e., % initial tension, α1: 46.8 ± 10.3%; α2: 39.0 ± 5.8%; α1 + α2: 48.7 ± 16.3% in the 10th set; all P < 0.05 vs. control). We conclude that sequential bouts of contractions induce a progressively greater degree of α-adrenergic receptor-induced arteriolar (but not venular) vasoconstriction which contributes significantly to fatigue in this model.     

Ser16-, but not Thr17-phosphorylation of phospholamban influences frequency-dependent force generation in human myocardium

-Adrenoceptor/cAMP-dependent Ser¹⁶-phosphoryation as well as Ca²⁺-dependent Thr¹⁷-phosphorylation of phospholamban (PLB) influences SERCA 2a activity and thus myocardial contractility. To determine the cross-signaling between Ca²⁺ and cAMP pathways, the phosphorylation of Ser¹⁶-PLB and Thr¹⁷-PLB was studied at increasing stimulation frequencies as well as in the presence of -adrenergic stimulation in isolated ventricular trabeculae from failing (dilative cardiomyopathy, DCM, heart transplants, n=9) and non-failing human myocardium (donor hearts, NF, n=9). In addition, we measured the intracellular Ca²⁺-transient (fura-2) at increasing stimulation frequencies (0.5–3.0 Hz). Protein expression of SERCA 2a and phospholamban was similar in DCM and NF. In DCM, diastolic [Ca²⁺]_i was increased and systolic [Ca²⁺]_i as well as Ser¹⁶ PLB-phosphorylation were decreased as compared to NF at 0.5 Hz. The positive force–frequency relationship in human non-failing myocardium was accompanied by a frequency-dependent increase in Ser¹⁶-PLB, but not Thr¹⁷-PLB phosphorylation. In DCM, Ser¹⁶-PLB as well as Thr¹⁷-PLB phosphorylation were not altered at higher stimulation frequencies. After application of isoprenaline (1 µM), a profound increase in Ser¹⁶-PLB phosphorylation was accompanied by a small increase in Thr¹⁷-PLB phosphorylation, only in NF. The frequency-dependent phosphorylation of Ser¹⁶-PLB may favor an increase in Ca²⁺ transient and force generation in humans. Cross talk signaling of Ser¹⁶/Thr¹⁷-PLB phosphorylation after -adrenergic stimulation exists in non-failing, but not in failing human myocardium. The Ca²⁺-dependent CaM-kinase activity may be altered in human heart failure.     

Potentiation of isometric and isotonic contractions during high-frequency stimulation

Activity dependent potentiation is an enhanced contractile response resulting from previous contractile activity. It has been proposed that even a maximal effort contraction may be enhanced by prior activity if there is an increase in the peak rate of force development. This should increase the peak active force during a very brief maximal effort contraction. The purpose of these experiments was to evaluate potentiation during brief sequential contractions with high-frequency stimulation. For this experiment, the rat medial gastrocnemius muscle was isolated in situ. Sequential stimulation (two contractions per second for 4 s) with 200, 300, or 400 Hz doublets, triplets, and quadruplets was applied. A small degree of force potentiation was observed in isometric contractions at the reference length (RL), but the activity dependent potentiation of isometric contractions was greater at short muscle length. For example, peak rate of force development for 200 Hz doublets increased significantly from the first to the eighth contraction (from 0.30 ± 0.02 to 0.34 ± 0.02 N·s⁻¹ at RL and from 0.18 ± 0.02 to 0.28 ± 0.01 N·s⁻¹ at RL-3 mm). During isotonic contractions, there were significant increases in peak shortening from the first to the eighth contraction. With 200 Hz doublet stimulation, shortening increased from 0.85 ± 0.14 to 1.14 ± 0.17 mm, and this corresponded with an increase in peak velocity (from 116 ± 18 to 136 ± 19 mm·s⁻¹). Remarkably, even 400 Hz quadruplets showed a significant increase in shortening during repeated contractions (2 s⁻¹). These observations indicate the possibility that activity dependent potentiation can result in significant improvement in both isometric and dynamic contractions, even when activated at very high frequency.     

Myofilament-based relaxant effect of isoprenaline revealed during work-loop contractions in rat cardiac trabeculae

In cardiac muscle, β-adrenergic stimulation increases contractile force and accelerates relaxation. The relaxant effect is thought to be due primarily to stimulation of Ca²⁺ uptake into the sarcoplasmic reticulum (SR), although changes in myofilament properties may also contribute. The present study investigated the contribution of the myofilaments to the β-adrenergic response in isolated rat cardiac trabeculae undergoing either isometric or work-loop contractions (involving simultaneous force generation and shortening) at different stimulation frequencies (range 0.25-4.5 Hz). SR-dependent effects were eliminated by treatment with ryanodine (1 μM) and cyclopiazonic acid (30 μM). In isometric contractions during SR inhibition, isoprenaline increased the force but did not alter the time course of the twitch. In contrast, in work-loop contractions, the positive inotropic effect was accompanied by a reduced diastolic force between beats, most apparent at higher frequencies (e.g. diastolic stress fell from 58.6 ± 5.5 to 28.8 ± 5.8 mN mm⁻² at 1.5 Hz). This relaxant effect contributed to a β-adrenoceptor-mediated increase in net work and power output at higher frequencies, by reducing the amount of work required to re-lengthen the muscle. Consequently, the frequency for maximum power output increased from 1.1 ± 0.1 to 1.6 ± 0.1 Hz. We conclude that the contribution of myofilament properties to the relaxant effect of β-stimulation may be of greater significance when force and length are changing simultaneously (as occurs in the heart) than during force development under isometric conditions.     

α1-Adrenoceptor agonists and IGF-1, myocardial hypertrophic factors, regulate the Kv1.5 K+ channel expression differentially in cultured newborn rat ventricular cells

 Interest has arisen concerning the importance of α-adrenergic function and insulin-like growth factor-1 (IGF-1) in cardiac remodelling. The hypothesis that these two factors may underlie the regulation of voltage-gated K⁺ channel expression in hypertrophied cardiomyocytes was tested by performing Western blot analysis of the Kv1.5 K⁺ channel α-subunit in cultured newborn rat ventricular cells. Myocyte size was quantified by surface area and total cell protein concentration. Cell exposure to the α₁-adrenoceptor agonist phenylephrine (PE, 20 μM) and IGF-1 (60 ng/ml) for 72 h both induced a significant increase of cell size indicating myocyte hypertrophy, which could be separately blocked by the protein kinase C inhibitor staurosporine (20 nM) and the tyrosine kinase inhibitor genistein (15 μM). Western blots of cell proteins prepared from myocyte cultures showed a single protein band at 75 kD recognized by the anti-Kv1.5 antibody, and demonstrated a 56% reduction in the Kv1.5 immunoreactive protein level in the PE-treated cell preparations. This suppression was not affected by staurosporine, but was remarkably attenuated by W7 (20 μM), a selective calmodulin antagonist. In contrast to PE, a 48% enhancement of the protein expression of Kv1.5 channel was induced by IGF-1 and this stimulation was specifically blocked by genistein. Our findings suggest that the differential regulation of cardiac Kv1.5 K⁺ channel expression can be produced by α₁-adrenoceptor activation and IGF-1 via distinctive signalling pathways. Calmodulin-dependent kinase and tyrosine kinase contribute importantly to the α₁-adrenoceptor-mediated decrease and the IGF-1-mediated increase in cardiac Kv1.5 K⁺ channel expression, respectively. Received: 19 August 1997 / Accepted: 14 January 1998     

Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery

K⁺-depolarization (KCl) of smooth muscle has long been known to cause Ca²⁺-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca²⁺ sensitization. This study examined effects of ROCK inhibition on the concentration–response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca²⁺ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 μM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca²⁺ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in β-escin permeabilized tissue. GF-109203X at 1 μM depressed the maximum Ca²⁺-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca²⁺ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 μM) strongly depression the Ca²⁺ CRCs in tissues permeabilized with Triton X-100, α-toxin and β-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca²⁺] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that β-escin permeabilized muscle preserves GPCR-independent, Ca²⁺- and ROCK-dependent, Ca²⁺ sensitization.     

甲亢大鼠心肌细胞缩短幅值-频率关系的变化

目的： 观测实验性甲状腺功能亢进症(甲亢)大鼠不同时期心功能变化的特征，以探明甲亢大鼠心力衰竭的可能机制。方法：采用大鼠无创尾套法测量血压与脉率，离体工作心脏模型观测心功能，利用离体单心肌细胞测量心肌收缩性能的变化。结果：实验性甲亢大鼠心率明显增快，心肌细胞宽度增加而长度未见明显改变。2周甲状腺素（T4）处理组离体工作心脏模式下，心输出量较对照组明显增强；在固有心率下，前负荷对心功能的调节机制保持正常。2周T4处理组心肌细胞无负荷缩短幅值，在1与2 Hz较对照组显著增加，但在4 Hz下与对照组无差别；随刺激频率增大，缩短幅值逐步降低。4周 T4组心肌细胞缩短幅值进一步增大，亦呈随频率增加而幅值降低的现象。两个T4组心肌细胞收缩时程均较对照组明显缩短。在1与2 Hz作用下，心肌细胞的无负荷缩短与舒张速率均明显高于对照组；4 Hz下2周T4组缩短与舒张速率仍高于对照组，4周T4组则与对照组间无显著差别。结论：4 周甲亢大鼠单心肌细胞无负荷缩短幅值-频率关系早于整体心脏功能发生变化。     

Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [³H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy.