199 Takotsubo syndrome associated mir-16 and mir-26a reduce contractility of cardiomyocytes in vitro by an inhibitory g-?protein dependent mechanism
199 Takotsubo syndrome associated mir-16 and mir-26a reduce contractility of cardiomyocytes in vitro by an inhibitory g-?protein dependent mechanism
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199 Takotsubo 综合征相关的 mir-16 和 mir-26a 通过抑制性 g-β 蛋白依赖性机制降低体外心肌细胞的收缩力
DOI：
10.1136/heartjnl-2017-311726.197
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发表时间：
2017
期刊：
影响因子：
5.7
通讯作者：
Couch L
中科院分区：
文献类型：
--
作者：
Couch L
简介 Takotsubo 综合征 (TTS) 是一种严重但可逆的急性心力衰竭，主要影响绝经后女性，其中心室心尖运动不能由压力引起的肾上腺素极度水平引起。多效性 β2AR 通过刺激性 (Gs) 和抑制性 (Gi) G 蛋白发出信号，虽然 Gs 增加心输出量，但同时也会降低存活率。 β2AR 的双重性是一种稳态机制，通过促进向 Gi 的转换来限制心脏毒性，尽管具有心脏抑制作用，但仍具有心脏保护作用。这在 TTS 中是失调的，过度的刺激会输送到 Gi，导致严重的负性肌力增强。目前尚不清楚患者易患 TTS 的原因是什么，但已经确定了 miR-16 和 miR-26a 增加的 microRNA (miR) 谱。鉴于 miR 在其他心脏疾病中的重要性以及 TTS 被认为与 β2AR-Gi 因果关系，我们假设这些 miR 可能导致 TTS 中的心脏抑制。方法使用 Lipofectamine 3000 在盲转染的成年大鼠心尖心肌细胞中操作 miR。使用 Ionoptix 系统测量缩短百分比，并应用药理学方案。使用 Fluo-4-AM 获得钙瞬态值，并通过微量咖啡因测量肌浆网 (SR) 钙含量。 N 数字显示为 n/N，其中 n/N= 细胞/大鼠。 结果 miR-16 和 miR-26a 的上调显着降低了基础收缩力（与对照组相比，miR-16=3.520.34%；n/N=30/6；p<0.05，miR-26a=2.770.21%）对照=4.300.43%；n/N=50/10；p<0.01），而下调则没有效果。 miR-16/-26a 操作不会改变 β2AR 反应。用百日咳毒素（PTX）抑制Gi可以防止这种情况（未处理的miR-16=5.080.49%，n=22；与经PTX处理的miR-16=8.820.63%，n=20；p<0.001；未经处理的miR-26a=3.200.29%相比经PTX处理的miR-26a=5.050.48%； n/N=30/6；p<0.05）。 PTX 处理没有改变对照转染细胞的收缩性。双重 miR-16/-26a 转染未观察到协同作用，这可能表明存在统一的机制。钙瞬变幅度随着 miR-16/-26a 上调而降低（对照的 F/F0=1.850.04，n/N=66/4 对比 miR-16=1.510.04，n/N=37/4 和 miR-26a=1.570.04，n/N=26/4；p<0.001），同时 SR 降低钙含量（对照的咖啡因诱导的 F/F0=3.130.14，n/N=32/4 对比 miR-16=2.130.13，n/N=22/4 和 miR-26a=2.570.19，n/N=17/4；分别为 p<0.001 和 p<0.05）。结论/含义增加miR-16/−26a 可能通过共同的 Gi 依赖机制在体外降低心肌细胞的基础收缩力。钙瞬变幅度的降低也可能有所贡献。这表明这些 miR 可能在机制上参与 TTS，但需要进一步的工作来研究它们的具体机制和时空参与。
IntroductionTakotsubo syndrome (TTS) is a severe but reversible acute heart failure affecting predominantly post-menopausal women, where ventricular apical akinesis results from extreme adrenaline levels arising with stress. The pleiotropic β²AR signals via stimulatory (Gs) and inhibitory (Gi) G-proteins, and whilst Gsincreases cardiac output, it concomitantly decreases survival. The duality of β²AR is a homeostatic mechanism to limit cardiotoxicity by facilitating a switch to Gi, serving as cardioprotective despite being cardiodepressive. This is dysregulated in TTS where excess stimulus trafficking to Giresults in profound negative inotropy. It is not understood what predisposes patients to TTS, but a microRNA (miR) profile of increased miR-16 and miR-26a has been identified. Given the importance of miRs in other cardiac diseases and that TTS is thought to be causally related to β2AR-Gi, we hypothesise that these miRs could predispose to the cardiodepression in TTS.MethodmiRs were manipulated in adult rat apical cardiomyocytes with blinded transfection using Lipofectamine 3000. Percentage shortening was measured using an Ionoptix system, and pharmacological protocols applied. Calcium transients were obtained using Fluo-4-AM and sarcoplasmic reticulum (SR) calcium content measured with caffeine micro-application. N numbers displayed as n/N, where n/N=cells/rats.ResultsUp-regulation of miR-16 and miR-26a significantly reduced basal contractility (miR-16=3.520.34% versus control=4.910.46%; n/N=30/6; p<0.05 and miR-26a=2.770.21% versus control=4.300.43%; n/N=50/10; p<0.01), whereas down-regulation had no effect. miR-16/-26a manipulation did not alter β2AR response. Inhibiting Gi with pertussis toxin (PTX) prevented this (miR-16 untreated=5.080.49%, n=22; versus miR-16 PTX-treated=8.820.63%, n=20; p<0.001; and miR-26a untreated=3.200.29% versus miR-26a PTX-treated=5.050.48%; n/N=30/6; p<0.05 respectively). PTX-treatment did not change contractility of control transfected cells. No synergism was observed with dual miR-16/-26a transfection possibly suggesting a unified mechanism. Calcium transient amplitude was decreased with miR-16/-26a up-regulation (F/F0 for control=1.850.04, n/N=66/4 versus miR-16=1.510.04, n/N=37/4 and miR-26a=1.570.04, n/N=26/4; p<0.001), along with a concomitant decrease in SR calcium content (caffeine-induced F/F0 for control=3.130.14, n/N=32/4 versus miR-16=2.130.13, n/N=22/4 and miR-26a=2.570.19, n/N=17/4; p<0.001 and p<0.05 respectively).Conclusion/implicationIncreased miR-16/−26a reduce basal contractility of cardiomyocytesin vitro, possibly through a shared Gi-dependent mechanism. Decreased calcium transient amplitude is also likely to contribute. This suggests these miRs may be mechanistically involved in TTS, but further work is needed to investigate their specific mechanistic and spatiotemporal involvement.