Centro de Investigaciones Cardiovasculares, CONICET La Plata, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 60 y 120, 1900, La Plata, Argentina.
Basic Res Cardiol. 2017 Dec 22;113(1):7. doi: 10.1007/s00395-017-0665-7. Print 2018 Jan 8.
In different pathological situations, cardiac cells undergo hyperosmotic stress (HS) and cell shrinkage. This change in cellular volume has been associated with contractile dysfunction and cell death. Given that nitric oxide (NO) is a well-recognized modulator of cardiac contractility and cell survival, we evaluated whether HS increases NO production and its impact on the negative inotropic effect observed during this type of stress. Superfusing cardiac myocytes with a hypertonic solution (HS: 440 mOsm) decreased cell volume and increased NO-sensitive DAF-FM fluorescence compared with myocytes superfused with an isotonic solution (IS: 309 mOsm). When cells were exposed to HS in addition to different inhibitors: L-NAME (NO synthase inhibitor), nitroguanidine (nNOS inhibitor), and Wortmannin (eNOS inhibitor) cell shrinkage occurred in the absence of NO release, suggesting that HS activates nNOS and eNOS. Consistently, western blot analysis demonstrated that maintaining cardiac myocytes in HS promotes phosphorylation and thus, activation of nNOS and eNOS compared to myocytes maintained in IS. HS-induced nNOS and eNOS activation and NO production were also prevented by AMPK inhibition with Dorsomorphin (DORSO). In addition, the HS-induced negative inotropic effect was exacerbated in the presence of either L-NAME, DORSO, ODQ (guanylate cyclase inhibitor), or KT5823 (PKG inhibitor), suggesting that NO provides contractile support via a cGMP/PKG-dependent mechanism. Our findings suggest a novel mechanism of AMPK-dependent NO release in cardiac myocytes with putative pathophysiological relevance determined, at least in part, by its capability to reduce the extent of contractile dysfunction associated with hyperosmotic stress.
在不同的病理情况下，心肌细胞会经历高渗应激（HS）和细胞收缩。这种细胞体积的变化与收缩功能障碍和细胞死亡有关。鉴于一氧化氮（NO）是心肌收缩力和细胞存活的公认调节剂，我们评估了高渗是否会增加 NO 的产生及其对这种应激下观察到的负性变力作用的影响。与用等渗溶液（IS：309 mOsm）灌流的心肌细胞相比，用高渗溶液（HS：440 mOsm）灌流会导致细胞体积减小并增加 NO 敏感的 DAF-FM 荧光。当细胞在 HS 存在的情况下暴露于不同的抑制剂时：L-NAME（NOS 抑制剂）、硝普胍（nNOS 抑制剂）和 Wortmannin（eNOS 抑制剂），细胞在没有 NO 释放的情况下发生收缩，表明 HS 激活了 nNOS 和 eNOS。Western blot 分析一致表明，与在 IS 中维持的心肌细胞相比，将心肌细胞维持在 HS 中会促进 nNOS 和 eNOS 的磷酸化，从而激活 nNOS 和 eNOS。HS 诱导的 nNOS 和 eNOS 激活和 NO 产生也被 Dorsomorphin（DORSO）抑制 AMPK 所阻止。此外，在存在 L-NAME、DORSO、ODQ（鸟苷酸环化酶抑制剂）或 KT5823（PKG 抑制剂）的情况下，HS 诱导的负性变力作用加剧，表明 NO 通过 cGMP/PKG 依赖性机制提供收缩支持。我们的研究结果表明，在心肌细胞中存在一种新型的 AMPK 依赖性 NO 释放机制，其潜在的病理生理相关性至少部分取决于其减少与高渗应激相关的收缩功能障碍程度的能力。
