Mechanism of preserved positive lusitropy by cAMP-dependent drugs in heart failure

In tachycardia-induced heart failure (HF), positive lusitropic effects of milrinone or dobutamine were assessed by evaluating the time constant of left ventricular (LV) pressure decay (tau) and Ca(2+)-ATPase activity of the sarcoplasmic reticulum (SR). The peak value of the positive first derivative of LV pressure (+dP/dt) was less increased, either by dobutamine (2-10 microg x kg(-1) x min(-1)) or by milrinone (4-20 microg/kg), in HF than in control (P < 0.05), whereas tau was shortened to an extent similar to that in control with dobutamine [P = not significant (NS)] and to an even greater extent with milrinone (P < 0.05). Ca(2+)-ATPase activity increased similarly in HF and control with dobutamine (1 microM; +11% in HF vs. +12% in control, P = NS), whereas it increased more with milrinone (1 microM; +19% in HF vs. +11% in control, P < 0.05). Ca(2+)-ATPase activity-cAMP relationships were shifted to the left by milrinone or dobutamine in HF compared with control. Thus, in HF, the sensitivity of Ca(2+)-ATPase activity to cAMP was increased on addition of cAMP-dependent inotropic agents, contributing to the preservation of positive lusitropy.     

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.     

肾上腺髓质素对大鼠损伤性心肌肌浆网功能的改善

通过观察下述五个指标,评价肾上腺髓质素(adrenomedullin,Adm)对大鼠损伤性心肌肌浆网功能的改善程度左心室压力最大变化速率(±dp/dtmax)、肌浆网钙摄取和释放及钙泵活性.皮下注射异丙肾上腺素(isoproterenol,ISO,69μmol/kg体重)制备大鼠心肌损伤坏死模型.摘取心脏后用Adm灌流,观察左心室压力最大变化速率(±dp/dtmax);制备并提纯心肌肌浆网(sarcoplasmicreticulum,SR)膜,测定SRCa2+摄取和释放速率、SR钙泵活性和钙通道蛋白～3H-ryanodine受体的最大结合量.结果发现,5×10-5mol/LAdm灌流能使ISO损伤的大鼠心脏左室±dp/dtmax分别增加16.9%(2?135±281vs1?980±302)和29.2%(1?375±267vs1?064±355,均P<0.05);SRCa2+摄取和释放率分别增加23.0%(15.0±1.4vs12.2±1.2)和43.5%(6.6±1.0vs4.6±0.6,均P<0.01);SRCa2+-ATPase活性和～3H-ryanodine受体最大结合量(Bmax)分别增加24.2%(P<0.01)和42.2%(P<0.05).提示Adm对ISO诱导的大鼠心肌损伤具有保护作用,其机制可能与Adm增加SRCa2+-ATPase活性、增加～3H-ryanodine所致SRCa2+摄取和释放升高有关.外源性给予Adm对损伤心肌可能具有临床治疗作用.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Cardiac dysfunction in terms of left ventricular mechanical work and energetics in hypothyroid rats

We hypothesized that cardiac dysfunction in hypothyroidism is mainly caused by the impairment of Ca(2+) handling in excitation-contraction coupling. To prove this hypothesis, we investigated left ventricular (LV) mechanical work and energetics without interference of preload and afterload in an excised, blood-perfused whole heart preparation from hypothyroid rats. We found that LV inotropism and lusitropism were significantly depressed, and these depressions were causally related to decreased myocardial oxygen consumption for Ca(2+) handling and for basal metabolism. The oxygen costs of LV contractility for Ca(2+) and for dobutamine in the hypothyroid rats did not differ from those in age-matched normal rats. The expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) significantly decreased and that of phospholamban significantly increased. The present results revealed that changes in LV energetics associated with decreased mechanical work in hypothyroid rats are mainly caused by the impairment of Ca(2+) uptake via SERCA2. We conclude that the impairment of Ca(2+) uptake plays an important role in the pathogenesis of cardiac dysfunction in hypothyroidism.     

Effect of vitamin C and L-NMMA on the inotropic response to dobutamine in patients with heart failure

The positive effect of vitamin C on left ventricular (LV) inotropic responses to dobutamine, observed in patients with preserved LV function, is lost in heart failure (HF). We tested the hypothesis that in HF, endogenous nitric oxide (NO) opposes the positive effect of vitamin C on adrenergically stimulated contractility by examining the effects of vitamin C on dobutamine responses during NO synthase inhibition. In 11 HF patients, a micromanometer-tipped catheter was inserted into the LV and an infusion catheter was positioned in the left main coronary artery. The peak positive rate of change of LV pressure (LV +dP/dt) was measured in response to intravenous dobutamine (Dob-1). After recontrol, intracoronary N(G)-monomethyl-L-arginine (l-NMMA) was infused before reinfusion of dobutamine (L-NMMA + Dob-2). Finally, intracoronary vitamin C was infused in addition to intracoronary L-NMMA and dobutamine (L-NMMA + Dob-2 + vitamin C). Intracoronary L-NMMA alone had no effect on LV +dP/dt. After a stable inotropic response to intracoronary L-NMMA and dobutamine was established, the addition of intracoronary vitamin C resulted in a modest but significant increase in LV +dP/dt. The change in LV +dP/dt in response to dobutamine alone was 25 +/- 5%, with intracoronary L-NMMA, 27 +/- 6%, and with intracoronary L-NMMA plus vitamin C, 37 +/- 5% (P < 0.05 vs. Dob-1 and L-NMMA + Dob-2). These findings demonstrate that an interaction between endogenous NO and redox environment exists and exerts some influence on stimulated contractility in HF.     

The effect of cAMP-dependent protein kinase phosphorylation on the external Ca2+ binding sites of cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum (SR) is known to be phosphorylated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase on a 22,000-dalton protein, Phosphorylation is associated with an increase in both the initial rate of Ca2+ uptake and the Ca(2+)-ATPase activity which is partially due to an increase in the affinity of the Ca(2+)-Mg(2+)-ATPase (E) of sarcoplasmic reticulum for calcium. In this study, the effect of cAMP-dependent protein kinase phosphorylation on the binding of calcium to the SR and on the dissociation of calcium from the SR was examined. The rate of dissociation of the E x Ca2 was measured directly and was not found to be significantly altered by cAMP-dependent protein kinase phosphorylation. Since the affinity of the enzyme for Ca2+ is equal to the ratio of the on and off rates of calcium, these results demonstrate that the observed change in affinity must be due to an increase in the rate of calcium binding to the Ca(2+)-Mg(2+)-ATPase of SR. In addition, an increase in the degree of positive cooperativity between the two calcium binding sites was associated with protein kinase phosphorylation.     

In situ SR function in postinfarction myocytes.

Previous studies have shown lower systolic intracellular Ca(2+) concentrations ([Ca(2+)](i)) and reduced sarcoplasmic reticulum (SR)-releasable Ca(2+) contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca(2+) entry via L-type Ca(2+) channels was normal, but that via reverse Na(+)/Ca(2+) exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca(2+) contents in MI myocytes, we measured SR Ca(2+) uptake and SR Ca(2+) leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca(2+) and inhibit SR Ca(2+) uptake resulted in a 10-fold prolongation of half-time (t(1/2)) of [Ca(2+)](i) transient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca(2+)](i) transient during a twitch in rat myocytes was primarily mediated by SR Ca(2+)-ATPase and that the t(1/2) of [Ca(2+)](i) decline is a measure of SR Ca(2+) uptake in situ. At 5.0 mM extracellular Ca(2+), systolic [Ca(2+)](i) was significantly (P 相似文献   

Effects of fatigue and training on sarcoplasmic reticulum Ca(2+) regulation in human skeletal muscle.

Little is known about fatigue and training effects on sarcoplasmic reticulum (SR) function in human muscle, and we therefore investigated this in eight untrained controls (UT), eight endurance-trained (ET), and eight resistance-trained athletes (RT). Muscle biopsies (vastus lateralis) taken at rest and after 50 maximal quadriceps contractions (180 degrees/s, 0.5 Hz) were analyzed for fiber composition, metabolites and maximal SR Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase activity. Fatigue reduced (P < 0.05) Ca(2+) release (42.1 +/- 3.8%, 43.4 +/- 3.9%, 31.3 +/- 6.1%), Ca(2+) uptake (43.0 +/- 5.2%, 34.1 +/- 4.6%, 28.4 +/- 2.8%), and Ca(2+)-ATPase activity (38.6 +/- 4.2%, 48.5 +/- 5.7%, 29.6 +/- 5.0%), in UT, RT, and ET, respectively. These decreases were correlated with fatigability and with type II fiber proportion (P < 0.05). Resting SR measures were correlated with type II proportion (r > or = 0.51, P < 0.05). ET had lower resting Ca(2+) release, Ca(2+) uptake, and Ca(2+)-ATPase (P < 0.05) than UT and RT (P < 0.05), probably because of their lower type II proportion; only minor effects were found in RT. Thus SR function is markedly depressed with fatigue in controls and in athletes, is dependent on fiber type, and appears to be minimally affected by chronic training status.     

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.     

Enhanced sarcoplasmic reticulum Ca(2+) release following intermittent sprint training

To evaluate the effect of intermittent sprint training on sarcoplasmic reticulum (SR) function, nine young men performed a 5 wk high-intensity intermittent bicycle training, and six served as controls. SR function was evaluated from resting vastus lateralis muscle biopsies, before and after the training period. Intermittent sprint performance (ten 8-s all-out periods alternating with 32-s recovery) was enhanced 12% (P < 0.01) after training. The 5-wk sprint training induced a significantly higher (P < 0.05) peak rate of AgNO(3)-stimulated Ca(2+) release from 709 (range 560-877; before) to 774 (596-977) arbitrary units Ca(2+). g protein(-1). min(-1) (after). The relative SR density of functional ryanodine receptors (RyR) remained unchanged after training; there was, however, a 48% (P < 0.05) increase in total number of RyR. No significant differences in Ca(2+) uptake rate and Ca(2+)-ATPase capacity were observed following the training, despite that the relative density of Ca(2+)-ATPase isoforms SERCA1 and SERCA2 had increased 41% and 55%, respectively (P < 0.05). These data suggest that high-intensity training induces an enhanced peak SR Ca(2+) release, due to an enhanced total volume of SR, whereas SR Ca(2+) sequestration function is not altered.     

不同频率电刺激膈神经导致隔肌肌浆网Ca^2＋—ATPase和Ca^2＋释放—摄取动力学改变

研究不同频率慢性电刺激（CES）后兔膈肌肌浆网（SR）Ca^2 -ATPase活性以及SRC^2 摄取－释放动力学对不同频率CES的活应性变化,建立不同频率CES组,用定磷法测定SR Ca^2 -ATPaes活性,用Fura-2荧光法测定SR Ca^2 摄取－释放动力学,与对照组比较,慢性低频电刺激10Hz和20Hz组的SR Ca^2 －ATPase活性明显降低（P＜0．01）,Ca^2 释放－摄动力学也显著降低（P＜0．01）,慢性高频电刺激50Hz和100Hz组的SRCa^2 -ATPase活性则显著升高（P＜0．01）,Ca^2 释放－摄取动力学亦明显升高（P＜0．01）,实验提示,ECS后不同频率CES导致膈肌SRCa^2 -ATPase,Ca^2 摄取－释放动力学产生不同的适应性变化,对不同功能状态的膈应用不同频谱的慢性电刺激可能具有重要的临床意义。     

Impaired sarcoplasmic reticulum Ca(2+) release rate after fatiguing stimulation in rat skeletal muscle.

The purpose of the study was to characterize the sarcoplasmic reticulum (SR) function and contractile properties before and during recovery from fatigue in the rat extensor digitorum longus muscle. Fatiguing contractions (60 Hz, 150 ms/s for 4 min) induced a reduction of the SR Ca(2+) release rate to 66% that persisted for 1 h, followed by a gradual recovery to 87% of prefatigue release rate at 3 h recovery. Tetanic force and rate of force development (+dF/dt) and relaxation (-dF/dt) were depressed by approximately 80% after stimulation. Recovery occurred in two phases: an initial phase, in which during the first 0.5-1 h the metabolic state recovered to resting levels, and a slow phase from 1-3 h characterized by a rather slow recovery of the mechanical properties. The recovery of SR Ca(2+) release rate was closely correlated to +dF/dt during the slow phase of recovery (r(2) = 0.51; P < 0.05). Despite a slowing of the relaxation rate, we did not find any significant alterations in the SR Ca(2+) uptake function. These data demonstrate that the Ca(2+) release mechanism of SR is sensitive to repetitive in vitro muscle contraction. Moreover, the results indicate that +dF/dt to some extent depends on the rate of Ca(2+) release during the slow phase of recovery.     

Effect of PDE5 inhibition on coronary hemodynamics in pacing-induced heart failure

Inhibition of phosphodiesterase type 5 (PDE5) can relax systemic and coronary vessels by causing accumulation of cGMP. Both the endothelial dysfunction with decreased nitric oxide production and increased natriuretic peptide levels in congestive heart failure (CHF) have the potential to alter cGMP production, thereby influencing the response to PDE5 inhibition. Consequently, this study examined the effects of PDE5 inhibition with sildenafil in dogs with CHF produced by rapid ventricular pacing. CHF resulted in decreases of left ventricular (LV) systolic pressure, coronary blood flow, and the maximal first time derivative of LV pressure (LV dP/dt(max)) at rest and during treadmill exercise compared with normal, whereas resting LV end-diastolic pressure increased from 10 +/- 1.4 to 23 +/- 1.4 mmHg. Sildenafil (2 and 10 mg/kg per os) caused a 5- to 6-mmHg decrease of aortic pressure (P < 0.05), with no change of heart rate, LV systolic pressure, or LV dP/dt(max). Sildenafil caused no change in coronary flow or myocardial oxygen consumption in animals with CHF at rest or during exercise. In contrast to findings in normal animals, sildenafil did not augment endothelium-dependent coronary vasodilation in response to acetylcholine in animals with CHF. Furthermore, Western blotting showed decreased PDE5 protein expression in myocardium from failing hearts. These findings demonstrate that PDE5 contributes little to regulation of coronary hemodynamics in CHF.     

Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 +/- 0.3 mo (n = 9)] and old rats [30.6 +/- 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 +/- 790 vs. 11,106 +/- 555 mmHg/s, P < 0.05) and isovolumic relaxation time (tau) was increased (8.7 +/- 0.7 vs. 6.3 +/- 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after beta-adrenergic receptor stimulation with dobutamine (20 mug/kg), which increased LV dP/dt by 170 +/- 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 +/- 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 +/- 0.2 vs. 1.3 +/- 0.2%), alpha-tubulin (92%), and beta-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins.     

Adenosine triphosphatases during maturation of cultured human skeletal muscle cells and in adult human muscle.

Na+/K(+)-ATPase, Mg(2+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase are examined in cultured human skeletal muscle cells of different maturation grade and in human skeletal muscle. Na+/K(+)-ATPase is investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase). SR Ca(2+)-ATPase is examined by ELISA, Ca(2+)-dependent phosphorylation and its activities on ATP and 3-O-methylfluorescein phosphate. Na+/K(+)-ATPase and SR Ca(2+)-ATPase are localized by immunocytochemistry. The activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase show a good correlation with the other assayed parameters of these ion pumps. All ATPase parameters investigated increase with the maturation grade of the cultured muscle cells. The number of ouabain-binding sites and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-MFPase are significantly higher in cultured muscle cells than in muscle. The Mg(2+)-ATPase activity, the content of SR Ca(2+)-ATPase and the activities of SR Ca(2+)-ATPase and Ca(2+)-dependent 3-O-MFPase remain significantly lower in cultured cells than in muscle. The ouabain-binding constant and the molecular activities of Na+/K(+)-ATPase and SR Ca(2+)-ATPase are equal in muscle and cultured cells. During ageing of human muscle the activity as well as the concentration of SR Ca(2+)-ATPase decrease. Thus the changes of the activities of the ATPases are caused by variations of the number of their molecules. Na+/K(+)-ATPase is localized in the periphery of fast- and slow-twitch muscle fibers and at the sarcomeric I-band. SR Ca(2+)-ATPase is predominantly confined to the I-band, whereas fast-twitch fibers are much more immunoreactive than slow-twitch fibers. The presence of cross-striation for Na+/K(+)-ATPase and SR Ca(2+)-ATPase in highly matured cultured muscle cells indicate the development and subcellular organization of a transverse tubular system and SR, respectively, which resembles the in vivo situation.