压力超负荷心肌肥大晚期和心衰大鼠心肌力学及Ca2+反应性的改变

目的 :观测比较肥大心肌和衰竭心肌在基础力学性能、[Ca2 ]o 正性变力作用和Ca2 通道阻滞剂负性变力作用三方面的变化和差别。方法 :大鼠升主动脉缩窄法建立左心室肥大 (LVH)和充血性心衰 (CHF)模型 ,记录离体灌流乳头肌的长度 张力曲线 ,比较基础状态和nifidipine处理后 [Ca2 ]o 与峰张力的量效关系。结果 :①CHF组长度 张力曲线较LVH组和假手术对照组 (Sham)明显下移 ;基础状态下 ([Ca2 ]o=0 .5mmo1/L) ,LVH等长收缩峰张力 (PT)尚能维持 ,但CHF组PT分别低于LVH组和Sham组 37.9%和 43 .7% (P均 <0 .0 1) ;±dT/dtmax也较LVH组进一步下降 (P <0 .0 1) ;②各组峰张力随 [Ca2 ]o 的升高而增强 ,但CHF组 [Ca2 ]o 作用的量效曲线较LVH和Sham明显下移 ;③nifedjpine的负性肌力作用具明显剂量依赖性 ,但其抑制程度在三组间没有明显差异 (P>0 .0 5 )。结论 :肥大心肌以收缩强度不变区别于衰竭心肌的收缩低抑。两组收缩速度、舒张速率和 [Ca2 ]o 正变力效应的下降程度递增 ,但Ca2 通道阻滞剂的负变力效应均无改变。     

E4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca^2＋水平的影响

目的：研究具有钠钙交换（NCX）激动作用的药物E 4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca2＋水平的影响。方法：通过腹主动脉缩窄建立大鼠慢性心力衰竭模型;利用Langendorff装置进行离体心脏灌流,检测大鼠心功能及E 4031对血流动力学指标的影响;急性分离心衰大鼠心肌细胞,与钙荧光指示剂fluo3/AM共同孵育后,用激光共聚焦显微镜系统观察E 4031对心肌细胞内荧光强度的影响。结果：缩窄大鼠腹主动脉12周后,langendorff离体灌流检测显示大鼠心功能明显降低;在灌流液中加入10μmol/L E 4031可以使心衰大鼠心脏左室发展压（LVDP）和左室收缩/舒张最大速率（±dp/dtmax）提高;与正常组和伪手术组相比,心衰大鼠心肌细胞内静息钙荧光强度明显升高,和10μmol/L E 4031共孵育后,心衰大鼠心肌细胞静息钙荧光强度呈现短期先升后降过程,然后在较低的水平保持稳定。结论：E 4031可以增强慢性心衰大鼠离体心功能,可能与其增强心肌细胞膜NCX活动,稳定细胞内Ca2＋水平有关。     

雌激素通过调节Gα_s-cAMP通路减轻异丙肾上腺素导致的大鼠心肌损伤

本研究旨在通过给予去卵巢大鼠异丙肾上腺素制作心肌损伤及心功能异常模型,探讨雌激素通过调节兴奋性G(Gαs)蛋白-环磷酸腺苷(cAMP)信号通路纠正儿茶酚胺导致的心功能异常的机制。观察雌激素对大鼠血流动力学参数:左心室收缩峰压(LVSP)、左心室舒张末压(LVEDP)、左心室内压上升的最大变化速率(+dp/dtmax)、左心室内压下降的最大变化速率(-dp/dtmax),血浆脑尿钠肽(brain natriuretic peptide,BNP),cAMP浓度和心肌中Gαs蛋白表达的影响。结果显示:与假手术组相比,去卵巢大鼠的血流动力学参数、血浆BNP水平、血浆cAMP水平没有显著改变;但给予去卵巢大鼠异丙肾上腺素后,血流动力学参数LVSP、+dp/dtmax降低(P0.01),LVEDP、-dp/dtmax升高(P0.01),血浆BNP水平升高(P0.01),血浆cAMP水平降低(P0.01);而进一步的雌激素补充则改善了心功能:LVSP、+dp/dtmax升高(P0.01),LVEDP、-dp/dtmax降低(P0.05,P0.01),血浆BNP水平降低(P0.01),cAMP水平升高(P0.01);雌激素对Gαs蛋白表达没有显著影响。结果提示:雌激素对心肌损伤具有保护作用,升高cAMP水平,改善心肌收缩过度抑制,调节心脏的功能状态。     

色胺酮对心肌缺血大鼠心功能和心肌组织COX-2蛋白表达等影响

目的:观察色胺酮对结扎冠状动脉所致心肌缺血大鼠心功能、心肌病理和心肌中COX-2的影响.方法采用结扎冠状动脉左前降支致心肌缺血模型,色胺酮0.10、0.20、0.30g/kg灌胃给药21天后,观察色胺酮对心肌缺血大鼠心功能、心肌病理和心肌组织COX-2蛋白的影响.结果①色胺酮0.10g/kg对心肌缺血大鼠的心脏功能有明显的改善作用,能增强心肌收缩力,改善心肌舒张的顺应性,表现为左心室内收缩压、左心室内压最大变化速率±dp/dtmax明显升高,与模型组比较有明显差异(P<0.05);②0.10g/kg色胺酮对结扎冠状动脉所致心肌缺血大鼠能明显减轻心肌变性,心肌纤维增生和淋巴细胞浸润;③色胺酮明显抑制缺血心肌组织中COX-2蛋白,其抑制作用随剂量的增大而逐渐增强.结论0.10g/kg色胺酮对大鼠结扎冠状动脉所致心肌缺血损伤具有明显的保护作用.     

心衰时心肌收缩蛋白分子基因表达和心肌收缩力的相互关系

目的和方法：本文通过ＤＯＣＡ硅胶管皮下埋入法建立大鼠心力衰竭模型,比较正常与心衰大鼠心肌收缩力的变化。采用ＲＮＡｓｌｏｔｂｌｏｔ杂交从基因转录水平检测正常与心衰大鼠心肌组织中心肌收缩蛋白分子基因αｃａｒｄｉａｃａｃｔｉｎ与αＭＨＣ表达的变化。结果：（１）心衰大鼠心肌收缩力较正常大鼠明显降低;（２）心衰大鼠与正常大鼠相比,心肌收缩蛋白分子基因αｃａｒｄｉａｃａｃｔｉｎ表达水平未见有统计学意义的变化,而αＭＨＣ表达水平呈显著降低（下降２１．３０％,Ｐ＜０．０５）;（３）αＭＨＣｍＲＮＡ的含量与心肌收缩力的大小存在线性正相关（ｒ＝０．４１４３,ｎ＝４３,Ｐ＜０．０５）。结论：αＭＨＣ基因表达水平的下降是心衰时心肌收缩力减退的主要分子基础之一,且与心肌收缩力的大小存在线性正相关     

类α-蝎神经毒素BmKⅠ对离体大鼠心脏收缩力及电活动的特异调控

本研究探讨了一种特异性钠通道调制剂(Buthus martensi Karsch,BmKⅠ)对离体大鼠心脏收缩力及电活动的调制作用.离体心脏灌流实验显示(1)BmKⅠ(0.5-10 μmol/L)剂量依赖地增强大鼠心肌收缩力,左心室最大发展压(LVDPmax)以及dp/dtmax与对照组相比均显著增强(n=6,P＜0.05),同时可触发正性变时作用(n=6,P＜0.05);(2)大剂量BmKⅠ(20μmol/L)引起负性肌力作用及心动过缓;(3)冠脉流量随心脏收缩力的增强反而减小,应用500nmol/L BmKⅠ时冠脉流量由14.5 ml/min降至8.6 ml/min(n=6,P＜0.05);此外,心电图记录表明BmKⅠ(0.5-10μmol/L)可触发心动过速及复杂的心律失常等电活动变化;正常灌流液洗脱后BmKI引起的大鼠心脏收缩力及电活动的改变可部分恢复.由于β-肾上腺素能受体阻滞剂普奈洛尔预先应用抑制了儿茶酚胺类神经递质的释放,提示BmKⅠ引起的大鼠心脏收缩力及电活动的改变不是由于其调节儿茶酚胺类神经递质的释放及随后β-肾上腺素能受体的激活,而可能与其对心肌电压门控钠通道的调控有关.     

安心酮对心血管系统作用的实验研究

目的研究安心酮（麻黄苯丙酮,Ｏｘｙｆｅｄｒｉｎｅ）对心脏血流动力学、冠脉流量和急性心肌缺血的药理效应方法观察药物对大鼠离体灌流心脏和在体狗心脏血流动力学、冠脉流量和心肌缺血的影响结果安心酮在一定的剂量范围内明显地增强心肌收缩力和对抗戊巴比妥所致的心力衰竭。但是,高剂量的安心酮可导致心功能抑制,使心肌收缩力和血压降低,β受体阻断剂可阻断其对心脏的激动作用,而安心酮又可拮抗异丙肾上腺素对心脏的兴奋作用。因此认为,本品可能是一种β受体部分激动剂。此外,安心酮可明显增加冠脉流量,减轻结扎冠状动脉引起的心肌梗死,以及拮抗高剂量的脑垂体后叶素所致的急性心肌缺血,这些作用可能是激动血管β２受体、选择性扩张冠脉和降低心肌耗氧量的结果结论安心酮明显地增强心肌收缩力、增加冠脉流量和改善心肌缺血。     

白介素-2对离体右心室乳头肌的作用及其机制

目的 :研究白介素 2 (IL 2 )对心肌跨膜电位和收缩作用的影响及其可能机制。方法 :在离体灌流大鼠右心室乳头肌标本上 ,采用常规细胞内玻璃微电极技术 ,观察IL 2 (0 .5～ 2 0 0u/ml)灌流 10min对心肌细胞跨膜电位的影响 ,并记录乳头肌收缩力变化。结果 :IL 2缩短心肌动作电位时程 (APD50 和APD80 ) ,但对静息电位、动作电位 0期去极化幅度及其速度无明显影响。IL 2呈剂量依赖性地抑制心肌收缩力 ,0 .5、2 .5、10、5 0和 2 0 0u/ml的IL 2分别使乳头肌收缩力下降至加药前的 94 .8%± 6 .8%、85 .8%± 6 .5 %、76 .3%± 7.8%、6 9.3%± 9.5 %和 5 2 .5 %±11.0 %。L NAME(10 -4mol/L)预处理 10min ,完全阻断较低浓度 (u/ml)IL 2 (0 .5、2 .5、10 )的心肌抑制作用 ,部分阻断高浓度IL 2 (5 0u/ml和 2 0 0u/ml)的作用。结论 :IL 2对离体右心室乳头肌的动作电位时程和收缩力有抑制作用 ,其负性变力作用与NO途径有关     

β2肾上腺素受体表达增加对心衰大鼠心肌细胞收缩功能的影响

目的：观察增加β2-肾上腺素受体（β2-AR）的表达对心衰大鼠心肌细胞收缩功能的影响并对其机制进行初步探讨：方法：用大剂量异丙肾上腺素制作大鼠心衰模型,分离培养心衰大鼠心肌细胞,在其上增加β2-AR的表达,westem blot检测心肌细胞β2-AR的表达。测定异丙肾上腺素刺激引起的细胞收缩幅度的改变：结果：心衰组心肌细胞收缩幅度较正常对照组降低（P〈0．01）,增加β2-AR表达可增加心衰组心肌细胞的收缩幅度（P〈0．01,心衰＋Adv．β2组vs心衰组）;选择性β2-AR拮抗剂ICI118,551可部分反转这种效应（P〈0．5,心衰＋Adv,β2＋ICI组讲心衰＋Adv．β2组）,但不能使收缩幅度降到心衰组水平（P〈0．05,心衰＋Adv．β2＋ICI组vs心衰组）.选择性β1—AR桔抗剂CGP20712A可完全阻断β2-AR表达增加的效应结论：增加β2-AR表达,可使心衰的心肌细胞的收缩功能得到改善,这种作用可能与β1-AR有关.     

乙酰胆碱洗脱后所致离体兔心的正性肌力作用

目的:乙酰胆碱(ACh)灌注并迅速洗脱后所致离体兔心正性肌力作用(心肌收缩力的反跳)的规律及其机制的初步研究.方法:采用Langendorff离体心脏灌流技术,观察对照组与实验组在给予ACh并洗脱后的心肌收缩力变化.结果:10-8～10-3 mol/L浓度的ACh所致心肌收缩力的最大反跳率分别为2.20%±1.70%、6.71%±3.40%、9.18%±3.54%、14.16%±3.27%、4.37%±5.86%、1.03%±6.86%;肾上腺素组的最大反跳率为29.25%±5.83%,普萘洛尔组最大反跳率为5.15%±4.45%,二者与对照组同浓度ACh(10-5mol/L)相比,均有显著统计学差异(P<0.05);维拉帕米组持续800 s未出现反跳现象.结论:在离体兔心,ACh洗脱后所致的正性肌力作用与心肌Ca2+通道、B受体的活性有关;ACh洗脱后与高浓度ACh时引起正性肌力作用的机制可能存在不同.     

The effect of insulin-like growth factor-1 on adult rat cardiac contractility

There is increasing evidence that insulin-like growth factor-1 (IGF-1) may play a role in both physiological and pathophysiological events in the mammalian myocardium. The present study investigated the acute effects of IGF-I on isometric force development in isolated rat cardiac muscle and on intracellular calcium (Ca²⁺) handling in isolated cardiac myocytes. IGF-I had a positive inotropic effect on rat ventricular papillary muscles increasing force development by 17.8 ± 4.6%, 18.5 ± 5.8% and 11.9 ± 4.9% (n = 12–20) at concentrations of 1, 10 and 100 ng/ml respectively. Isoprenaline increased tension in these papillary muscles by 56.7 ± 7.7% at a concentration of 100 nM (n = 22). In comparison, insulin increased papillary muscle force development by 11.6 ± 3.2%, 17.7 ± 4.1% and 19.7 ± 5.6% at concentrations of 1, 10 and 100 nM respectively (n = 16–20). In the single cardiac myocyte IGF-1 increased, the peak cytosolic free Ca²⁺ concentration, the amplitude of the Ca²⁺ transient and the time to peak Ca²⁺ as measured with the fluorescent bioprobe Indo-1 AM. The positive inotropic response to IGF-1 by rat ventricular muscle is therefore associated with a rise in free, peak cytosolic Ca²⁺ in isolated cardiac myocytes. Increasing insulin concentrations (1–1000 nM) elicited a progressive elevation in isometric force and free, cytosolic Ca²⁺. In contrast, in the presence of IGF-1, the maximal rise in isometric force and free cytosolic Ca²⁺ were both observed at 10 ng/ml. Recent reports have suggested that IGF-1 may act on the mammalian myocardium when administered chronically, but this study is amongst the first to demonstrate an acute effect of IGF-I on the mammalian heart. IGF-1 may prove then to be a novel cardioactive agent in both normal and pathophysiological states.     

Cyclic GMP and cyclic AMP induced changes in control and hypertrophic cardiac myocyte function interact through cyclic GMP affected cyclic-AMP phosphodiesterases.

We tested the hypothesis that the negative functional effects of cyclic GMP (cGMP) would be greater after increasing cyclic AMP (cAMP), because of the action of cGMP-affected cAMP phosphodiesterases in cardiac myocytes and that this effect would be altered in left ventricular hypertrophy (LVH) produced by aortic valve plication. Myocyte shortening data were collected using a video edge detector, and O2 consumption was measured by O2 electrodes during stimulation (5 ms, 1 Hz, in 2 mM Ca2+) from control (n = 7) and LVH (n = 7) dog ventricular myocytes. cAMP and cGMP were determined by a competitive binding assay. cAMP was increased by forskolin and milrinone (10(-6) M). cGMP was increased with zaprinast and decreased by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxilin-1-one (ODQ) both at 10(-6) and 10(-4) M, with and without forskolin or forskolin + milrinone. Zaprinast significantly decreased percent shortening in control (9 +/- 1 to 7 +/- 1%) and LVH (10 +/- 1 to 7 +/- 1%) myocytes. It increased cGMP in control (36 +/- 5 to 52 +/- 7 fmol/10(5) myocytes) and from the significantly higher baseline value in LVH (71 +/- 12 to 104 +/- 18 fmol/10(5) myocytes). ODQ increased myocyte function and decreased cGMP levels in control and LVH myocytes. Forskolin + milrinone increased cAMP levels in control (6 +/- 1 to 15 +/- 2 pmol/10(5) myocytes) and LVH (8 +/- 1 to 18 +/- 2 pmol/10(5) myocytes) myocytes, as did forskolin alone. They also significantly increased percent shortening. There were significant negative functional effects of zaprinast after forskolin + milrinone in control (15 +/- 2 to 9 +/- 1%), which were greater than zaprinast alone, and LVH (12 +/- 1 to 9 +/- 1%). This was associated with an increase in cGMP and a reduction in the increased cAMP induced by forskolin or milrinone. ODQ did not further increase function after forskolin or milrinone in control myocytes, despite lowering cGMP. However, it prevented the forskolin and milrinone induced increase in cAMP. In hypertrophy, ODQ lowered cGMP and increased function after forskolin. ODQ did not affect cAMP after forskolin and milrinone in LVH. Thus, the level of cGMP was inversely correlated with myocyte function. When cAMP levels were elevated, cGMP was still inversely correlated with myocyte function. This was, in part, related to alterations in cAMP. The interaction between cGMP and cAMP was altered in LVH myocytes.     

Milrinone inhibits contractility in skinned skeletal muscle fibers

Direct action of the cardiotonic bipyridine milrinone on thecross bridges of single fibers of skinned rabbit skeletal muscle wasinvestigated. At 10°C and pH 7.0, milrinone reduced isometric tension in a logarithmically concentration-dependent manner, with a55% reduction in force at 0.6 mM. Milrinone also reducedCa²⁺ sensitivity of skinned fibersin terms of force production; the shift in the force-pCa curveindicated a change in the pCa value at 50% maximal force from 6.10 to5.94. The unloaded velocity of shortening was reduced by 18% in thepresence of 0.6 mM milrinone. Parts of the transient tension responseto step change in length were altered by milrinone, so that the testand control transients could not be superimposed. The results indicatethat milrinone interferes with the cross-bridge cycle and possiblydetains cross bridges in low-force states. The results also suggestthat the positive inotropic effect of milrinone on cardiac muscle isprobably not due to the drug's direct action on the muscle crossbridges. The specific and reversible action of the bipyridine on muscle cross bridges makes it a potentially useful tool for probing the chemomechanical cross-bridge cycle.

     

Reduction in interaction between cGMP and cAMP in dog ventricular myocytes with hypertrophic failure

Baseline function and signal transduction are depressed in hearts with hypertrophic failure. We tested the hypothesis that the effects of cGMP and its interaction with cAMP would be reduced in cardiac myocytes from hypertrophic failing hearts. Ventricular myocytes were isolated from control dogs, dogs with aortic valve stenosis hypertrophy, and dogs with pacing hypertrophic failure. Myocyte function was measured using a video edge detector. Cell contraction data were obtained at baseline, with 8-bromo-cGMP (10(-7), 10(-6), and 10(-5) M), with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA; a cAMP phosphodiesterase (PDE(2)) inhibitor] plus 8-bromo-cGMP, or milrinone (a PDE(3) inhibitor) plus 8-bromo-cGMP. Baseline percent shortening and maximal rates of shortening (R(max)) and relaxation were slightly reduced in hypertrophic myocytes and were significantly lower in failing myocytes (R(max): control dogs, 95.3 +/- 17.3; hypertrophy dogs, 88.2 +/- 5.5; failure dogs, 53.2 +/- 6.4 mum/s). 8-Bromo-cGMP dose dependently reduced myocyte function in all groups. However, EHNA (10(-6) M) and milrinone (10(-6) M) significantly reduced the negative effects of cGMP on cell contractility in control and hypertrophy but not in failing myocytes (R(max) for control dogs: cGMP, -46%; +EHNA, -21%; +milrinone, -19%; for hypertrophy dogs: cGMP, -40%; +EHNA, -13%; +milrinone, -20%; for failure dogs: cGMP, -40%; +EHNA, -29%; +milrinone, -32%). Both combinations of EHNA-cGMP and milrinone-cGMP significantly increased intracellular cAMP in control, hypertrophic, and failing myocytes. These data indicated that the cGMP signaling pathway was preserved in hypertrophic failing cardiac myocytes. However, the interaction of cGMP with the cAMP signaling pathway was impaired in these failing myocytes.     

Augmentation of the inotropic response to insulin in diabetic rat hearts.

Insulin participates in the modulation of myocardial function, but its inotropic action in diabetes mellitus is not fully clear. In the present study, we examined contractile responses to insulin in left-ventricular papillary muscles and ventricular myocytes isolated from hearts of normal or short-term (5-7 days) streptozotocin-induced (65 mg/kg) diabetic rats. Mechanical properties of papillary muscles and ventricular myocytes were evaluated using a force transducer and an edge-detector, respectively. Contractile properties of papillary muscles or cardiac myocytes, electrically stimulated at 0.5 Hz, were analyzed in terms of peak tension development (PTD) or peak twitch amplitude (PTA), time-to-peak contraction (TPT) and time-to-90% relaxation (RT90). Intracellular Ca2+ transients were measured as fura-2 fluorescence intensity change (deltaFFI). Insulin (1-500 nM) had no effect on PTD in normal myocardium, whereas it produced a positive inotropic response in preparations from diabetic animals, with a maximal increase of 11%. Insulin did not modify TPT or RT90 in either group. Further studies revealed that insulin enhanced cell shortening in diabetic but not normal myocytes, with a maximal increase of 21%. Consistent with its action on the mechanical properties of papillary muscles and cardiac myocytes, insulin also induced a dose-dependent increase in the intracellular Ca2+ transient in diabetic but not normal myocytes. Collectively, these data suggest that the myocardial contractile response to insulin may be altered in diabetes.     

Cellular distribution of GPR14 and the positive inotropic role of urotensin II in the myocardium in adult rat.

Urotensin II is a cyclic neuropeptide recently shown to play a role via its receptor GPR14 in regulating vascular tone in the mammalian cardiovascular system. The existence of GPR14 in rat heart has been validated by ligand binding assay and RT-PCR. In the present study, we investigated the cellular distribution of GPR14 protein in rat heart by using immunohistochemistry and confocal microscopic immunofluorescence double staining with antipeptide polyclonal antibodies against GPR14 and cell type markers for myocytes and endothelial cells. The direct effect of urotensin II on left ventricular contractility was further evaluated in isolated left ventricular papillary muscles of the rat. In paraffin-embedded heart sections, positive immunohistochemical staining was observed in the left ventricle but not in the right ventricle and atria. Immunofluorescence double staining revealed the cardiac myocyte as the only cell type expressing GPR14 protein in frozen heart sections as well as in isolated cardiac myocytes. There was no visible signal for GPR14 in intramyocardial coronary arteries and capillaries. The existence of GPR14 protein in rat heart was further validated by immunoprecipitation and Western blot analysis. In isolated rat left ventricular papillary muscle preparations, urotensin II induced an increase in active contractile force. GPR14 mRNA was also detected in rat heart by RT-PCR. These data provide the first direct evidence for the cellular localization of GPR14 receptor protein and a positive inotropic effect of urotensin II in normal rat heart.     

cAMP and in vitro inotropic actions of secretin and VIP in rat papillary muscle.

Secretin and VIP stimulate cardiac adenylyl cyclase activity and exert a positive inotropic action in several mammalian species. This study examined positive inotropic activity and cAMP levels in rat papillary muscle. Isoproterenol and secretin increased contractions by 150+/-31% and 129+/-27%, respectively. VIP increased contraction by 30+/-21% only at 10 microM. Isoproterenol significantly increased cAMP levels by 82%, whereas increases by secretin (58%) and VIP (56%) were not significant. These results are consistent with reports that secretin and VIP stimulate cardiac adenylyl cyclase in the rat, but suggest that cAMP tissue levels cannot totally explain the positive inotropic responses to secretin and VIP.     

Nitric oxide-dependent and -independent mechanisms are involved in TNF-alpha -induced depression of cardiac myocyte contractility

Previous studies have demonstrated the presence of myocardial depression in clinical and experimental septic shock. This response is mediated, in part, through circulating TNF-alpha-induced, nitric oxide-dependent, depression of basal myocyte contractility. Other mechanisms of early myocardial dysfunction involving decreased response to adrenergic stimulation may exist. This study evaluated the presence and nitric oxide dependence of impaired adrenergic response to TNF-alpha in in vitro cardiac myocytes. The contraction of electrically paced neonatal rat cardiac myocytes in tissue culture was quantified using a closed-loop video tracking system. TNF-alpha induced depression of baseline contractility over the first 20 min of cardiac myocyte exposure. This effect was blocked by N-methyl-arginine (NMA), a nitric oxide synthase inhibitor, in all studies. Contractile and cAMP response to increasing concentrations of isoproterenol was deficient in cardiac myocytes exposed to TNF-alpha regardless of the presence of NMA. In contrast, increasing concentrations of forskolin (a direct stimulant of adenylate cyclase) and dibutyryl cAMP (a metabolically active membrane-soluble analog of cAMP) completely reversed TNF-alpha-mediated depression, though only in the presence of NMA. Forskolin-stimulated cAMP generation remained intact regardless of NMA. Increasing concentrations of exogenous calcium chloride, unlike other inotropic agents, corrected TNF-alpha-mediated defects of contractility independent of the presence of NMA. These data suggest that TNF-alpha exposure is associated with a second nitric oxide-independent but calcium-dependent early depressant mechanism that is manifested by reduced contractile and cAMP response to beta-adrenergic stimulation.     

Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes

A technique is presented that allows neonatal rat cardiac myocytes to form spontaneously and coherently beating 3-dimensional engineered heart tissue (EHT) in vitro, either as a plane biconcaval matrix anchored at both sides on Velcro-coated silicone tubes or as a ring. Contractile activity was monitored in standard organ baths or continuously in a CO(2) incubator for up to 18 days (=26 days after casting). Long-term measurements showed an increase in force between days 8 and 18 after casting and stable forces thereafter. At day 10, the twitch amplitude (TA) of electrically paced EHTs (average length x width x thickness, 11 x 6 x 0.4 mm) was 0.51 mN at length of maximal force development (L(max)) and a maximally effective calcium concentration. EHTs showed typical features of neonatal rat heart: a positive force-length and a negative force-frequency relation, high sensitivity to calcium (EC(50) 0.24 mM), modest positive inotropic (increase in TA by 46%) and pronounced positive lusitropic effect of isoprenaline (decrease in twitch duration by 21%). Both effects of isoprenaline were sensitive to the muscarinic receptor agonist carbachol in a pertussis toxin-sensitive manner. Adenovirus-mediated gene transfer of beta-galactosidase into EHTs reached 100% efficiency. In summary, EHTs retain many of the physiological characteristics of rat cardiac tissue and allow efficient gene transfer with subsequent force measurement.     

Effects of epidermal growth factor on epinephrine-stimulated heart function in rodents

Epidermal growth factor (EGF) interferes with beta-adrenergic receptor (beta-AR) signaling in adipocytes and hepatocytes, which leads to decreased lipolytic and glycogenolytic responses, respectively. We studied the effect of EGF on the heart. EGF interfered with the cAMP signal generated by beta-AR agonists in cardiac myocytes. In perfused hearts, EGF decreased inotropic and chronotropic responses to epinephrine but not to 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Sustained epinephrine infusion induced heart contracture, which resulted in altered heart function as demonstrated by decreased inotropy and increased heart rate variability. EGF prevented all these alterations. In the whole animal (anesthetized mice), EGF administration reduced the rise in heart rate induced by a single epinephrine dose and the occurrence of Bezold-Jarisch reflex episodes induced by repeated doses. Sialoadenectomy enhanced the response to epinephrine, and EGF administration restored normal response. All these results suggest that, by interfering with beta-AR signaling, EGF protects the heart against the harmful effects of epinephrine.