CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Comparison of Frontal EMG Biofeedback and Several Types of Relaxation Instructions in Reducing Multiple Indices of Arousal

During the training phase, 96 subjects were given one of four types of relaxation instructions (single instructions, repeated instructions, relaxation training, no instructions) and in addition either did or did not receive frontal EMG biofeedback training. Results indicated that each of the instructions and biofeedback procedures were equally effective in reducing frontal EMG, but that none of these procedures had any effect on subjective anxiety or autonomic indices of arousal (pulse rate, skin temperature, and finger pulse volume). During the generalization/stress phase, subjects were threatened with electric shock and were told to apply the relaxation techniques they learned during the training phase even though no additional instructions and/ or biofeedback training would be provided. To assess the effectiveness of the shock manipulation, a no-threat control group was included. Results indicated that: a) the shock manipulation was effective in increasing arousal, b) previous instructions and/or biofeedback were equally effective in reducing frontal EMG levels, but that c) only relaxation training was consistently effective in reducing subjective and autonomic indices of arousal. These findings: a) suggest that in stressful situations, relaxation training may be more effective than either EMG biofeedback or simple relaxation instructions in producing a general relaxation effect as opposed to a specific EMG effect; and b) indicate the importance of assessing the effectiveness of relaxation procedures during stressful situations during which subjects’ levels of arousal are elevated above resting baseline levels.     

“阶梯式倒U形”生物束带阴道紧缩术

目的介绍生物束带(脱细胞异体真皮,HADM)"阶梯式倒U形"阴道紧缩术的临床效果和操作方法。方法:从本手术项目组2018年1月至2019年9月期间实施的"阶梯式倒U形"生物束带阴道紧缩术案例中,随机选择50例作为研究对象;均由同一手术项目组按照统一操作规范,采用生物束带实施"阶梯式倒U形"阴道紧缩术。统计平均手术时间;观察术前、术后(即刻)阴道口及阴道前1/3大小,计算阴道收紧有效率;测量术前、术后(即刻)会阴体高度,计算会阴体抬高有效率;检测术后2月阴道握持力,计算阴道握持力增加有效率;观察术后并发症及其发生率;随访术后2月、6月、1年患者对手术满意度。结果:50例"阶梯式倒U形"生物束带阴道紧缩术平均手术时间28min;48例术后阴道明显收紧,有效率达96%;38例会阴体高度明显抬高,有效率达76%;45例术后2月阴道握持力明显增加,有效率达90%;术后均未发生排异反应,无直肠损伤、血肿、感染等。30例得到有效随访,随访率达60%;术后2月、6月、1年患者满意度分别为88%、97%、98%。结论:"阶梯式倒U形"生物束带阴道紧缩术手术操作快捷,可有效收紧阴道、修复会阴体和增加阴道握持力等,具有临床效果确切、并发症少、安全性高、可操作性强和求美者满意度高等优势,值得临床推广应用。     

Evaluation of the effect of Benson's relaxation technique on pain and quality of life of haemodialysis patients: A randomized controlled trial

Background

Haemodialysis patients may suffer from pain and impairment of quality of life. Some complementary interventions, such as relaxation therapy, might affect the pain and quality of life. The present study aimed to identify the effectiveness of Benson's relaxation technique in relieving pain and improving the quality of life in haemodialysis patients.

Study design

The study was a randomized controlled trial.

Setting and participants

The data were collected in two haemodialysis units affiliated to Shiraz University of Medical Sciences. A total of 86 haemodialysis patients were randomly assigned to either the intervention (receiving Benson's relaxation technique) or the control group (routine care) from 2011 to 2012.

Intervention

The patients in the intervention groups listened to the audiotape of relaxation technique twice a day each time for 20 min for eight weeks.

Measurements and outcomes

The pain numeric rating scale and Ferrans and Powers Quality of Life Index-dialysis version questionnaire were completed at baseline and 8 weeks after the intervention. The data were analyzed using independent t-test and ANCOVA.

Results

The results of ANCOVA showed a significant difference between the intervention and the control group concerning the mean score of the intensity of pain (F = 6.03, p = 0.01). Moreover, a significant difference was found between the intervention and the control group regarding the total quality of life (F = 10.20, p = 0.002) and health-functioning (F = 8.64, p = 0.004), socioeconomic (F = 12.45, p = 0.001), and family (F = 8.52, p = 0.005) subscales of quality of life.

Conclusion

These findings indicated that Benson's relaxation technique might relieve the intensity of pain and improve the quality of life in haemodialysis patients. Thus, Benson's relaxation technique could be used as part of the care practice for relieving the pain intensity and improvement of the quality of life in haemodialysis patients.     

放松训练改善门诊慢性病患者的焦虑情绪和睡眠质量

目的 探讨减轻门诊慢性病患者焦虑情绪、改善睡眠质量的简单有效的护理方法.方法 将60例在门诊接受输液治疗并存在焦虑情绪和睡眠障碍的慢性病患者随机分为实验组和对照组各30例,对照组患者单纯按医嘱实施常规输液治疗,实验组在常规输液治疗的基础上给予放松训练干预,疗程4周,采用焦虑自评量表(SAS)和睡眠自测量表(AIS)分别于干预前后对患者进行测评.结果 干预后两组患者焦虑评分和睡眠评分比较,实验组明显低于对照组,差异有统计学意义(P＜0. 01),实验组的焦虑程度和睡眠障碍较对照组明显减轻.结论 放松训练可以改善门诊慢性病患者的焦虑情绪和睡眠质量.     

Phospholamban is required for CaMKII-dependent recovery of Ca transients and SR Ca reuptake during acidosis in cardiac myocytes

Initially during acidosis, Ca transient amplitude (Delta[Ca]i) and the rate constant of [Ca]i decline (k(Ca)) are decreased, but later during acidosis Delta[Ca]i and k(Ca) partially recover. This recovery in rat myocytes could be inhibited by KN-93 suggesting that CaMKII-dependent protein phosphorylation (and enhanced SR Ca uptake) may be responsible. To test whether phospholamban (PLB) is required for the Delta[Ca]i and k(Ca) recovery during acidosis, we used isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. [Ca]i was measured using fluo-3. During the initial phase of acidosis (1-4 min), Delta[Ca]i decreased in WT myocytes (n = 8) from 1.75 +/- 0.19 to 1.10 +/- 0.13 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 3.20 +/- 0.22 to 2.38 +/- 0.18 s(-1) (P < 0.05). Later during acidosis (6-12 min), Delta[Ca]i partially recovered to 1.41 +/- 0.18 DeltaF/F0 and k(Ca) to 2.78 +/- 0.22 s(-1) (i.e. both recovered by approximately 50%). CaMKII inhibition using KN-93 completely prevented this recovery of Delta[Ca]i and k(Ca) during late acidosis in WT myocytes. In PLB-KO myocytes (n = 11) Delta[Ca]i decreased during early acidosis from 2.92 +/- 0.31 to 1.33 +/- 0.17 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 10.45 +/- 0.56 to 7.58 +/- 0.68 s(-1) (P < 0.05). However, Delta[Ca]i did not recover during late acidosis and k(Ca) decreased even more (6.59 +/- 0.65 s(-1)). Parallel results were seen for contractile parameters. We conclude that PLB is crucial to the recovery of Delta[Ca]i and k(Ca) during acidosis. Moreover, PLB phosphorylation by CaMKII plays an important role in limiting the decline in Ca transients (and contraction) during acidosis.     

Bronchorelaxation of the human bronchi by CFTR activators

The airway functions are profoundly affected in many diseases including asthma, COPD and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR (Cystic Fibrosis transmembrane Conductance Regulator) gene, which normally encodes a multifunctional and integral membrane cAMP regulated and ATP gated Cl⁻ channel expressed in airway epithelial cells.Using human lung tissues obtained from patients undergoing surgery for lung cancer, we demonstrated that CFTR participates in bronchorelaxation. Using human bronchial smooth muscle cells (HBSMC), we applied iodide influx assay to analyze the CFTR-dependent ionic transport and immunofluorescence technique to localize CFTR proteins. Moreover, the relaxation was studied in isolated human bronchial segments after pre-contraction with carbachol to determine the implication of CFTR in bronchodilation.We found in HBSMC that the pharmacology and regulation of CFTR is similar to that of its epithelial counterpart both for activation (using forskolin/genistein or a benzo[c]quinolizinium derivative) and for inhibition (CFTR_inh-172 and GPinh5a). With human bronchial rings, we observed that whatever the compound used including salbutamol, the activation of muscular CFTR leads to a bronchodilation after constriction with carbachol.Altogether, these observations revealed that CFTR in the human airways is expressed in bronchial smooth muscle cells and can be pharmacologically manipulated leading to the hypothesis that this ionic channel could contribute to bronchodilation in human.     

Frequency-dependent acceleration of relaxation in the heart depends on CaMKII,but not phospholamban

Frequency-dependent acceleration of relaxation (FDAR) is an intrinsic physiological mechanism, which allows more rapid ventricular diastolic filling at higher heart rates. FDAR is also observed in isolated myocardial trabeculae and cardiac myocytes, but its mechanism is still poorly understood. We tested the hypothesis that FDAR results mainly from Ca/calmodulin-dependent protein kinase II (CaMKII) dependent stimulation of sarcoplasmic reticulum (SR) Ca transport, but does not require phospholamban. Experiments were performed at 23 or 35 degrees C in isolated ventricular muscle and single myocytes from wild-type (WT) and phospholamban knockout (PLB-KO) mice and rat ventricular myocytes. Isometric twitch force of muscles and unloaded shortening and Ca transients in myocytes were measured ([Ca](o)=1mM) in the absence and presence of CaMKII inhibitors (1 microM KN-93 or 20 microM autocamtide-2 related inhibitory peptide, AIP). Stimulation frequency was altered over a wide range (0.2-8Hz) and post-rest vs steady state twitches were also compared. In both WT and PLB-KO mouse muscles FDAR of twitch force was prominent, but was largely suppressed by KN-93. FDAR of twitch contractions was associated with FDAR of Ca transients in PLB-KO myocytes, and both were inhibited by KN-93. Similarly, a different CaMKII inhibitor (AIP) inhibited FDAR of contraction and Ca transients in rat ventricular myocytes. We conclude that FDAR results mainly from CaMKII-dependent stimulation of SR Ca transport, but does not require phospholamban.     

色满类化合物HEF-19对家兔离体肠管平滑肌的舒张作用

目的:探讨色满类化合物HEF-19对家兔离体小肠平滑肌的舒张作用及其机制.方法:采用家兔小肠离体实验法, 观察HEF-19对家兔离体小肠自发活动的影响, 研究BaCl2诱导收缩时格列苯脲(10-5 mol/L)、维拉帕米(10-7 mol/L )、酚妥拉明(10-7 mol/L)和普奈洛尔(10-7 mol/L)对HEF-19的舒张作用的影响.结果:HEF-19可以剂量依赖性地降低家兔离体小肠平滑肌自发性收缩的张力和振幅; 分别加入格列苯脲和维拉帕米孵育后,HEF-19(10-6 mol/L-10-3 mol/L)的舒张量效曲线均发生明显变化, EC50值分别由4.36±0.12mol/L和4.43±0.19 mol/L转变为4.16±0.22mol/L和3.96±0.24 mol/L, 差异具有统计学意义(P <0.05). 酚妥拉明和普奈洛尔对HEF-19的舒张作用没有影响.结论:HEF-19具有舒张小肠平滑肌的作用并且其作用机制与ATP-敏感性钾通道和L 型钙通道开放有关.而与α受体和β受体无关.     

对氧磷对血管内皮细胞的损伤作用及机制探讨

目的为探讨有机磷酸酯对血管内皮细胞和血管内皮功能是否有直接的损伤作用。方法用不同浓度的对氧磷分别与大鼠离体血管环和培养的人脐静脉单层内皮细胞共孵不同的时间,以乙酰胆碱引起的血管内皮依赖性舒张反应和单层内皮细胞通透性等为观察指标,检测对氧磷对血管内皮的损伤作用。结果对氧磷（36.3nmol/L-36.3μmol/L）与血管环共孵,呈浓度和时间依赖性地显著抑制乙酰胆碱诱导的内皮依赖性舒张反应,而对硝普钠引起的非内皮依赖性舒张反应没有明显的影响。对氧磷与内皮细胞共孵不同的时间,呈浓度和时间依赖性地显著增加单层内皮细胞的通透性。对氧磷在损伤血管内皮的同时也导致了血管组织及细胞培养液中一氧化氮浓度和超氧化物歧化酶活性的降低、脂质过氧化代谢产物丙二醛浓度的升高。加入左旋精氨酸能部分拮抗对氧磷对血管内皮功能的损伤作用,用阿托品预处理则不影响对氧磷的作用。结论该研究提示对氧磷对血管内皮细胞有直接损伤作用,其机制可能与对氧磷诱发氧化应激,进而导致脂质过氧化反应发生和增加内皮细胞的通透性有关。