Laboratory of Vascular Physiology, Department of Biomedical and Chemical Engineering and Sciences, Florida Institute of Technology, Melbourne, USA.
Department of Physiology, Augusta University, Augusta, USA.
Sci Rep. 2021 Jan 14;11(1):1420. doi: 10.1038/s41598-021-80966-6.
Heat-shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone with various biological functions. Recently, we demonstrated that HSP70 is key for adequate vascular reactivity. However, the specific mechanisms targeted by HSP70 to assist in this process remain elusive. Since there is a wealth of evidence connecting HSP70 to calcium ([Formula: see text]), a master regulator of contraction, we designed this study to investigate whether blockade of HSP70 disrupts vascular contraction via impairment of [Formula: see text] handling mechanisms. We performed functional studies in aortas isolated from male Sprague Dawley rats in the presence or absence of exogenous [Formula: see text], and we determined the effects of VER155008, an inhibitor of HSP70, on [Formula: see text] handling as well as key mechanisms that regulate vascular contraction. Changes in the intracellular concentration of [Formula: see text] were measured with a biochemical assay kit. We report that blockade of HSP70 leads to [Formula: see text] mishandling in aorta stimulated with phenylephrine, decreasing both phasic and tonic contractions. Importantly, in [Formula: see text] free Krebs' solution, inhibition of HSP70 only reduced the [Formula: see text] of the phasic contraction if the protein was blocked before IP3r-mediated [Formula: see text] release, suggesting that HSP70 has a positive effect towards this receptor. Corroborating this statement, VER155008 did not potentiate an IP3r inhibitor's outcomes, even with partial blockade. In another set of experiments, the inhibition of HSP70 attenuated the amplitude of the tonic contraction independently of the moment VER155008 was added to the chamber (i.e., whether it was before or after IP3r-mediated phasic contraction). More compelling, following re-addition of [Formula: see text], VER155008 amplified the inhibitory effects of a voltage-dependent [Formula: see text] channel blocker, but not of a voltage-independent [Formula: see text] channel inhibitor, indicating that HSP70 has a positive impact on the latter. Lastly, the mechanism by which HSP70 modulates vascular contraction does not involve the [Formula: see text] sensitizer protein, Rho-kinase, nor the SERCA pump, as blockade of these proteins in the presence of VER155008 almost abolished contraction. In summary, our findings shed light on the processes targeted by HSP70 during vascular contraction and open research avenues for potential new mechanisms in vascular diseases.
热休克蛋白 70（HSP70）是一种普遍表达的分子伴侣，具有多种生物学功能。最近，我们证明 HSP70 是血管反应性的关键。然而，HSP70 协助这一过程的具体机制仍不清楚。由于有大量证据表明 HSP70 与钙（[Formula: see text]）有关，钙是收缩的主要调节剂，因此我们设计了这项研究来探讨 HSP70 的阻断是否通过损害钙处理机制来破坏血管收缩。我们在雄性 Sprague Dawley 大鼠的主动脉中进行了功能研究，这些主动脉存在或不存在外源性钙，我们还确定了 HSP70 抑制剂 VER155008 对钙处理以及调节血管收缩的关键机制的影响。通过生化测定试剂盒测量细胞内钙浓度的变化。我们报告说，HSP70 的阻断导致去甲肾上腺素刺激的主动脉钙处理不当，减少了相位和紧张性收缩。重要的是，如果在 IP3r 介导的钙释放之前阻断 HSP70，在无钙 Krebs 溶液中，HSP70 的抑制仅减少了相位收缩的钙，这表明 HSP70 对该受体有积极影响。这一说法得到了证实，即使部分阻断，VER155008 也不会增强 IP3r 抑制剂的效果。在另一组实验中，HSP70 的抑制作用独立于 VER155008 加入腔室的时刻（即在 IP3r 介导的相收缩之前或之后）减弱了紧张性收缩的幅度。更有说服力的是，在重新加入钙后，VER155008 放大了电压依赖性钙通道阻滞剂的抑制作用，但不能放大电压不依赖性钙通道抑制剂的抑制作用，表明 HSP70 对后者有积极影响。最后，HSP70 调节血管收缩的机制不涉及钙敏感受体蛋白、Rho 激酶或 SERCA 泵，因为在 VER155008 存在的情况下阻断这些蛋白几乎完全消除了收缩。总之，我们的发现揭示了 HSP70 在血管收缩过程中靶向的过程，并为血管疾病的潜在新机制开辟了研究途径。
