Sarcoplasmic reticulum Ca 2+ load in human heart failure

Excitation-contraction coupling and intracellular Ca ²⁺ homeostasis are altered in heart failure. We tested the hypothesis that these changes are related to disturbed Ca ²⁺ handling of the sarcoplasmic reticulum (SR). Isolated, electrically stimulated trabeculae were obtained from end-stage failing (NYHA IV) and nonfailing human hearts. Isometric twitch tension, intracellular Ca ²⁺ transients (aequorin method) and SR Ca ²⁺ content (rapid cooling contractures) were assessed under basal conditions (1 Hz, 37 °C) as well as after stepwise increasing rest intervals from 2 – 240 s (post-rest contractions). Protein expression of SERCA2a and phospholamban (Western blot) was assessed in a subset of failing trabeculae. In addition, the effects of SERCA1 overexpression on contractile function of isolated myocytes was tested. On average, post-rest twitch tension continuously increased with increasing rest intervals in nonfailing, but declined with rest intervals longer than 15s in failing myocardium. The rest-dependent contractile changes were accompanied by parallel changes in intracellular Ca ²⁺ transients. Failing trabeculae (n = 40) were grouped (group A: post-rest potentiation (force of contraction > pre-rest twitch force) after 120s rest interval; group B: post-rest decay (force of contraction < pre-rest twitch force) after 120 s rest interval), and post-rest contractile function was related to SERCA2a and PLB expression. While PLB protein expression was not different, SERCA2a protein expression as well as SERCA2a/PLB ratio was significantly higher in group A vs. group B. Transfection of SERCA1 increased shortening amplitude and enhanced relaxation kinetics in failing human myocytes. In conclusion, SR Ca ²⁺ handling is severely altered in human heart failure. Reduced SR Ca ²⁺ release is due to diminished SR Ca ²⁺ content directly related to a depressed expression of SERCA2a protein. Enhancing SERCA function or expression may improve SR Ca ²⁺ handling in failing human myocardium.     

Targeting Ca 2+ cycling proteins and the action potential in heart failure by gene transfer

Cardiomyocytes isolated from failing human hearts are characterized by contractile dysfunction including prolonged relaxation, reduced systolic force and elevated diastolic force. These contractile abnormalities are paralleled by abnormal Ca ²⁺ homeostasis such as reduced sarcoplasmic reticulum (SR) Ca ²⁺ release, elevated diastolic Ca ²⁺ and reduced rate of Ca ²⁺ removal. In addition, failing human myocardium is characterized by a frequency-dependent decrease in systolic force and Ca ²⁺ as opposed to normal myocardium where an increase in pacing rate results in potentiation of contractility and an increase in SR Ca ²⁺ release. In the failing heart, the decrease in SR Ca ²⁺ load has been linked to a decrease in SR Ca ²⁺ ATPase (SERCA2a) function. We have recently shown that overexpression of SERCA2a by adenoviral gene transfer restores contractile function in cardiac myocytes from failing human hearts. In addition, we have shown that overexpression of SERCA2a in a model of pressure-overload hypertrophy in transition to failure improves contractile function and reserve in these animals. We are currently exploring the effect of long-term expression of SERCA2a in failing animals along with the energy cost of SERCA2a expression using NMR methods. We are also using a different strategy to improve SR Ca ²⁺ ATPase activity which involves decreasing the expression of phospholamban by antisense strategies to enhance SR Ca ²⁺ ATPase activity. The Na/Ca exchanger is also being targeted to enhance calcium removal in failing hearts. Action potential prolongation is attributed to reductions in transient outward current (Ito) density in human heart failure. This prolongation can alter contractility but can also cause afterdepolarization. Using gene transfer of various K channels responsible for Ito, we are investigating the molecular and the ionic basis of action potential prolongation in cardiac hypertrophy and failure and we are examining how intracellular calcium handling changes in response to alterations in action potential duration. Gene transfer, which serves initially as an experimental tool, may provide a novel therapeutic approach.     

Voltage-gated Ca 2+ currents in the human pathophysiologic heart: a review

The L-type Ca ²⁺ current (I _Ca-L) plays a key role in the cardiac excitation-contraction (E-C) coupling. Thus, it is a major target for many transmitters and hormones modulating cardiac function and, therefore, for pharmacological drugs to regulate inotropy. Ca ²⁺ (and other) ion currents are commonly studied in animal tissues for practical reasons. Investigations in human cardiomyocytes started extensively only ten years ago with the development of patch-clamp techniques, enzymatic cell dissociation procedures, and surgical techniques. These studies have already provided valuable information concerning the nature, biophysics, pharmacology and regulation of human cardiac ionic currents in normal and diseased tissues. Interesting advances have been made to understand the role of I _Ca-L in the development of chronic atrial fibrillation (AF). Alterations of single channel activity and regulation of macroscopic I _Ca-L have also been found in heart failure (HF), although some of the data are divergent and puzzling. The T-type Ca ²⁺ current (I _Ca-T) has never been recorded in human cardiomyocytes. After a rapid overview of the basic properties of human cardiac Ca ²⁺ currents, we focus on selected aspects of pathophysiology that are still unsolved.     

普鲁卡因胺、利多卡因和普罗帕酮对豚鼠心室肌细胞Na^＋／Ca^2＋交换电流的抑制作用

目的利用膜片钳技术观察Ⅰ类抗心律失常药物普鲁卡因胺、利多卡因、普罗帕酮对Na+/Ca2+交换电流的直接作用.方法采用胶原酶消化的成年大鼠单个心室肌细胞及全细胞膜片钳技术,记录Na+/Ca2+交换电流并观察药物对它的影响.结果3种药物对Na+/Ca2+交换电流的抑制均呈剂量依赖性,但抑制程度不同,其中普鲁卡因胺抑制作用最强.50、100μM的普鲁卡因胺分别使外向Na+/Ca2+交换电流从对照值(181±22)pA降低至(125±19)、(109±20)pA,内向电流由对照值(172±18)pA分别降低至(137±13)、(121±12)pA;50、100μM的利多卡因使外向电流从对照值(170±15)pA分别降低至(139±15)、(127±10)pA,内向电流由对照值(165±15)p/A分别降至(142±16)、(129±20)pA;50、100μM的普罗帕酮使外向电流由对照值(160±23)pA分别降至(130±27)、(112±26)pA,内向电流由对照值(169±13)pA分别降至(143±13)、(134±14)pA.普鲁卡因胺、普罗帕酮对外向电流的抑制大于内向电流,而利多卡因对内、外向电流的抑制差异无显著性.结论Ⅰ类抗心律失常药物对心室肌细胞Na+/Ca2+交换电流具有直接抑制作用,且抑制程度不同.     

Increased Ca 2+-sensitivity of myofibrillar tension in heart failure and its functional implication

In human failing myocardium, an increased Ca ²⁺-sensitivity of myofilament tension development has been described in Triton X skinned cardiac myocytes compared to cardiomyocytes obtained from non-failing human donor hearts. The present study aimed to investigate whether there are functional implications of the increased Ca ²⁺-sensitivity in heart failure and whether alterations of myofilament function are already obvious at earlier stages of heart failure, such as in cardiac hypertrophy or whether alterations of the intracellular Ca ²⁺-homeostasis are able to induce alterations in myofilament function. Ca ²⁺-activated tension development was measured in Triton X-skinned fibers from human failing and non-failing myocardium. Ca ²⁺-sensitivity of myofilament tension development was significantly shifted to the left in human failing myocardium. Plots of diastolic free Ca ²⁺ versus diastolic tension development showed that in a range of similar diastolic Ca ²⁺-concentrations, diastolic tension was significantly enhanced in the failing hearts. The Ca ²⁺/tension relationship was shifted to the right in Triton X-skinned fiber preparations from transgenic renin overexpressing rats (TG(mREN2)27), shown to have concentric hypertrophy. In addition, the Ca ²⁺/tension relationship was unchanged in phospholamban knock-out mice with an increased systolic Ca ²⁺ (and enhanced diastolic Ca ²⁺-load). It is concluded that the increased Ca ²⁺-sensitivity of myofilament tension observed in single cardiomyocytes from failing human myocardium may be a phenomenon also present in multicellular preparations and may contribute to the diastolic dysfunction observed in human heart failure. Alterations of myofilament function occur at very early stages of heart failure and may be species dependent, or dependent on intracellular free Ca ²⁺-levels.     

COPD患者CD4+/CD8+、CD8+CD28+ T淋巴细胞检测意义

目的分析慢性阻塞性肺疾病(COPD)患者CD4⁺/CD8⁺、CD8⁺CD28⁺调节性T淋巴细胞检测意义。 方法收集2018年6月至2019年6月于我院就诊收治的98例COPD患者的临床资料,随访18个月,根据患者生存情况分为存活组85例与死亡组13例,采用流式细胞术检测COPD患者CD4⁺/CD8⁺、CD8⁺CD28⁺调节性T淋巴细胞,应用ROC曲线分析CD4⁺/CD8⁺及CD8⁺CD28⁺调节性T淋巴细胞预测COPD患者死亡的价值。 结果两组患者PaO₂、FEV₁占预测值、有创机械通气例数所占比、CD4⁺/CD8⁺以及CD8⁺CD28⁺ T淋巴细胞占比存在统计学差异(P<0.05);多因素Logistic回归分析,PaO₂、FEV₁占预测值、有创机械通气、CD4⁺/CD8⁺、CD8⁺CD28⁺是COPD预后的独立危险因素(P<0.05);CD4⁺/CD8⁺、CD8⁺CD28⁺ T淋巴细胞水平预测COPD患者死亡的最佳截断值分别为1.76%、7.85%,二者联合预测COPD患者死亡的ROC曲线下面积(AUC)为0.913(95%CI: 0.862~0.947),明显高于单项CD4⁺/CD8⁺的AUC值(0.784, 95%CI: 0.602~0.850)及CD8⁺CD28⁺的AUC值(0.795,95%CI：0.596~0.861)。 结论COPD患者CD4⁺/CD8⁺、CD8⁺CD28⁺ T淋巴细胞与患者预后密切相关,二者联合检测对COPD患者预后有意义。     

Na^＋／H^＋交换、Na^＋／K^＋／2Cl^－转运体抑制剂对缺血大鼠心脏保护作用的研究

目的研究Na+/H+交换抑制剂阿米洛利(Amiloride)、Na+/K+/2Cl-协同转运抑制剂呋塞米(Furosemide)对长时间低温保存下离体大鼠心脏的保护作用.方法建立Langendorff及工作心脏灌注模型.实验组用St.Thomas-2(STH-2)液+阿米洛利+呋塞米停搏并保存其中,对照组只用STH-2停搏、保存.置7 C环境5 h后恢复灌流.观察血流动力学、心肌酶学及超微结构的变化.结果实验组冠状动脉流量(CAF)、左心室收缩压(LVSP)、左心室压力变化速率(±dp/dt)的恢复率均优于对照组(P＜0.01);肌酸磷酸激酶(CPK)、乳酸脱氢酶(LDH)漏出量明显少于对照组(P＜0.05);心肌组织中Na+-K+-ATP酶、Ca2+-ATP酶和Ca2+-Mg2+-ATP酶活力均显著高于对照组(P＜0.01);实验组的心肌细胞超微结构得到较好的保护.结论Na+/H+交换、Na+/K+/2Cl-协同转运抑制剂对缺血大鼠心肌具有明显保护作用.     

Altered regulation of cardiac contraction and relaxation by Ca2+ in heart failure

Myocardial contractile dysfunction in congestive heart failure is characterized by a decrease in force developed and retardation of relaxation. These alterations are mainly due to those in intracellular Ca(2 +) transients (CaT) . CaT are regulated by a number of functional proteins, including sarcolemmal L-type Ca(2 +) channels, Na(+)/Ca(2 +) exchanger and Ca(2 +) ATPase, sarcoplasmic reticulum Ca(2 +) ATPase (SERCA 2 ) , phospholamban and ryanodine receptors, and mitochondrial Ca(2 +) uniporter. Changes in expression and function of these regulatory proteins that occur in the course of increasing severity of heart failure are responsible for the characteristic changes in force development and relaxation observed under pathophysiological conditions in congestive heart failure.     

CD4CD4+和CD8CD4+ T淋巴细胞与动脉粥样硬化相关性研究进展

在动脉粥样硬化的发生发展过程中CD4+和CD8+T淋巴细胞有着重要的意义,近年来越来越多的研究显示了CD4+和CD8+T淋巴细胞参与了动脉粥样硬化的免疫炎症反应,受到了国内外学者的关注,同时,也成为心血管领域和免疫学界难以攻关的课题。本文对CD4+和CD8+T淋巴细胞与动脉粥样硬化相关性研究进展做一简要综述。     

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase alpha2-isoform in heart failure

AIMS: The Na+/K+-ATPase (NKA) alpha2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca(+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA alpha2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction. METHODS AND RESULTS: NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (I NKA and I NCX) were measured and alpha2-isoform current (I NKA,alpha2) was separated from total I NKA using 0.3 microM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of alpha2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA alpha1- and alpha2-isoforms. I NKA,alpha2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When I NKA,alpha2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of I NKA led to a decrease in I NCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the alpha2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the alpha2-isoform is necessary to modulate local [Na+]i close to NCX. CONCLUSION: Downregulation of the alpha2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.     

Ca2+-handling in heart failure – a review focusing on Ca2+ sparks

[Ca ²⁺] _i-transients have been shown to be altered in isolated ventricular myocytes from terminally failing human myocardium. It has been demonstrated that one reason for this alteration is a reduction in the Ca ²⁺ content of the sarcoplasmic reticulum (SR). Further investigations were done to investigate, whether there may be an additional defect of the Ca ²⁺-release mechanisms from the SR. These release mechanisms were investigated through the recording of Ca ²⁺ sparks in single human myocytes. In cardiac myocytes, Ca ²⁺ sparks are elementary units of Ca ²⁺ release, which occur spontaneously, or which are triggered by Ca ²⁺ influx through L-type Ca ²⁺-channels (Ca ²⁺-induced Ca ²⁺ release). Ca ²⁺ sparks have been investigated in various animal models of cardiac hypertrophy and cardiac failure and results were conflicting. Discrepancies may be explained by different species and also by the mechanisms underlying hypertrophy and heart failure. This review summarizes our current knowledge on Ca ²⁺ sparks in heart failure.     

心脏再同步化治疗对缺血性心力衰竭犬的疗效及心肌钙通道电流的影响

目的研究心脏再同步化治疗(CRT)对失同步化缺血性心力衰竭犬的疗效及钙离子通道电流(ICa)的影响。方法取西北犬行左束支射频消融术和冠状动脉前降支结扎术建立失同步化缺血性心力衰竭模型(n=14)。随机选取7只造模成功犬行CRT治疗,作为CRT组。另7只造模成功犬作为失同步化组。取5只正常犬作为对照组。CRT后6周(其它两组于相应时间点)检测心电图学、超声心动图及血流动力学指标,上述指标检测完毕后,分离左室侧壁和前壁心肌细胞,用全细胞膜片钳法检测ICa。结果与对照组相比较,失同步化组的RR间期缩短(420±55 ms vs 598±98 ms,P<0.05),QTc间期延长(433±46 ms vs 378±32 ms,P<0.05),QRS波时限延长(100±23 ms vs 53±8 ms,P<0.05),失同步化指数(DI)升高(80±22 ms vs 28±6 ms,P<0.05)。与失同步化组比较,CRT组QRS波时限缩短(73±11 ms vs 100±23 ms,P<0.05),降低了DI(32±24 ms vs 80±22 ms,P<0.05),缩短了QTc间期(392±36 ms vs 433±46 ms,P<0.05)。对照组左室前壁和侧壁的ICa密度峰值无差别(-4.7±0.3pA/pF vs-4.5±0.3 pA/pF,P>0.05)。失同步化组侧壁ICa电流密度低于前壁(-3.8±0.2 pA/pF vs-5.2±0.3 pA/pF,P<0.01)。CRT组侧壁和前壁的电流密度无差别(-4.5±0.2 pA/pF vs-4.5±0.3 pA/pF,P>0.05)。结论 CRT能部分地逆转失同步化缺血性心力衰竭的电重构与机械重构。     

Kupffer细胞对日本血吸虫病肉芽肿期CD4~+CD25~+T细胞的影响

目的探讨Kupffer细胞对日本血吸虫病肉芽肿期CD4+CD25+T细胞的影响。方法6～8周龄雌性C57BL/6J小鼠30只分为对照组、感染组与感染/氯化钆组3组,每组10只。感染组和感染/氯化钆组小鼠通过腹部感染尾蚴(10条/只),感染/氯化钆组于感染后第4周经尾静脉注射氯化钆,剂量为每次15mg/kg,每周2次;对照组通过尾静脉注射PBS。感染8周后流式细胞仪检测小鼠CD4+CD25+T细胞数量;免疫组织化学染色检测Foxp3的分布;ELISA检测血清细胞因子IL-4、IL-5、IL-10、TGF-β1与IFN-γ的水平,并进行肝功能检测。结果感染组小鼠CD4+CD25+T细胞数量为13.8%,感染/氯化钆组为9.3%;感染组IL-10为41.4pg/ml,感染/氯化钆组为22.6pg/ml;氯化钆可下调Foxp3的分布、血清丙氨酸氨基转移酶的水平,并减轻血吸虫肉芽肿周围的炎症反应。结论Kupffer细胞通过调控CD4+CD25+T细胞数量而参与日本血吸虫肉芽肿的形成。     

Hyperinsulinaemia and Na+, K+ -ATPase activity in thyrotoxic periodic paralysis

OBJECTIVE Thyrotoxic periodic paralysis (TPP) usually follows a heavy carbohydrate meal and this may be explained by hyperinsulinaemia stimulating Na⁺, K⁺ -ATPase activity. To clarify this the effect of glucose load on serum insulin concentration and platelet Na⁺, K⁺ -ATPase activity In thyrotoxic periodic paralysis (TPP) was examined. DESIGN In all subjects a standard 75-g glucose tolerance test was done and blood samples were taken at 0, 1 and 2 hours. SUBJECTS Twenty-five healthy controls (8 M and 17 F), 17 uncomplicated thyrotoxic patients (7M and 10 F), 15 TPP patients who presented with paralysis and 4 TPP patients after treatment with antithyrold drugs. MEASUREMENTS Plasma glucose was measured by the glucose oxidase method, serum insulin by radioimmunoassay and platelet Na⁺, K⁺ -ATPase by the release of phosphate from ATP. RESULTS TPP patients showed glucose intolerance (area under the curve (AUC) 16·5 ± 4·4 (mean ± SD) In TPP compared to 12·9 ± 4·5 In controls (P < 0·01) and hyperinsulinaemia (AUC 189·6 ±100·6 vs 98·5 ±53·4, P < 0·001). In uncomplicated thyrotoxicosis the results were similar to that in healthy controls. Platelet Na⁺, K⁺ -ATPase were significantly higher in thyrotoxic patients compared to controls and In TPP patients were even higher. Ingestion of glucose increased platelet Na⁺, K⁺ -ATPase in all groups. AUC for platelet Na⁺, K⁺ -ATPase in TPP patients were significantly higher than in uncomplicated thyrotoxicosis (601 ±99·3 vs 482 ± 109·4, P < 0·01) or healthy controls (320 ± 107·3). In the 4 TPP patients studied after antithyroid treatment the results were similar to healthy controls. CONCLUSION Patients with thyrotoxic periodic paralysis have hyperinsulinaemia and this is accompanied by higher Na⁺, K⁺-ATPase activity.     

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

In the present study, we questioned whether the cholesterol synthesis inhibitor lovastatin potentiates the cytotoxicity of chemotherapeutic agents in the primitive CD34⁺ subpopulation of acute myeloid leukemia (AML) cells. AML mononuclear cells (n = 17) were sorted in CD34⁺ and CD34⁻ fractions and compared to normal CD34^+/− cells (n = 7). The percentage of surviving cells upon exposure to lovastatin (25–100 μM) and/or chemotherapeutics (cytarabin or daunorubicin) was determined with a luminescent cell viability assay. The results demonstrate that the primitive CD34⁺ subpopulation of normal and AML cells displayed a higher sensitivity to lovastatin than the more mature CD34⁻ subpopulation. The combination of lovastatin and chemotherapeutics resulted in a more pronounced inhibitory effect on both subpopulations. In contrast to the homogeneous results in normal CD34⁺ cells, a distinct heterogeneity in lovastatin sensitivity was found in AML samples. Therefore, a group of normal (n = 11) and abnormal (n = 6) responders were identified based on a reduced or increased cell survival compared to normal CD34⁺ cells. This distinction was not only observed in the CD34⁺ AML subfraction but also in CD34⁺CD38⁻AML cells. In the abnormal responder group, 50% of patients presented with unfavorable cytogenetics and significant higher peripheral blast cell counts, which coincided with poor treatment results. In summary, the findings indicate that the primitive subfraction of CD34⁺ AML cells is in the majority of cases affected by lovastatin treatment, which is potentiated when combined with chemotherapeutics. Heterogeneity of the response observed in AML patients allowed identification of a subgroup with poor prognosis.