Skeletal Muscle Function under Pressure Elucidating Mechanisms of Muscle Ca2+ Signaling Failure and Ultrastructure Disintegration using a novel High-Pressure Multiphoton Microscopy Technology

高压(HP)几乎影响所有的生物过程。对于我们地球上的大多数生物圈来说，长期的高静水压力暴露是规则，而不是例外。要了解HPS对活细胞或整个生物体的影响，需要有控制的压力条件和观察诱导变化的能力，以了解生物体如何抵消或适应增加的压力环境。第一项任务在技术上具有挑战性，例如，将实验密封起来，以抵御高达数百兆帕的环境压力梯度。第二项任务需要将HP条件与光学技术相结合，在细胞和组织的生物物理研究中将其作为显微镜技术。活细胞显微镜已经发展到非常先进的水平，然而，幽门螺杆菌很少被解决。从这样的HP实验中，可以获得其他生物物理技术所不能获得的针对高压应激的活细胞的分子反应。我们的目标是将显微镜和工程专业知识结合起来，开发一种新的HP血管，适用于HP下活细胞的多光子成像。这种新颖的光学室将被设计成能够承受高达400兆帕的压力，这代表了真核和原核细胞感兴趣的广泛压力范围。我们将把新的光学梯度折射率透镜技术直接应用到压力容器中，而不是使用玻璃窗和外部透镜。然后，该系统将被应用于研究(I)场刺激的哺乳动物骨骼肌细胞中的钙瞬变和(Ii)静止细胞中在从大气到深海环境的不同环境压力分布下的静止钙水平以及“压力X暴露时间”产品。这将揭示对压力引起的骨骼肌兴奋偶联钙稳态变化的新的生物物理学见解。利用肌球蛋白的二次谐波产生显微镜，我们将确定肌肉细胞肌节解体的压力阈值和动力学。这种方法可以研究压力引起的骨骼肌器官损伤的具体机制，这可以解释哺乳动物的主要暴露限值。建立的新显微技术将提供一种新的工具，将当代显微镜扩展到新的压力视界，并对不同类型细胞的生物物理过程进行光学研究，这将为许多不同复杂性的生物体的高压生物物理提供新的场所。

High Pressure (HP) affects virtually all biological processes. Prolonged high hydrostatic pressure exposures are the rule rather than the exception for most of our earths biosphere. Understanding HPs impact on living cells or whole organisms requires controlled pressure conditions and the ability to observe the induced changes in order to understand how organisms counteract or adapt to increased pressure environments. The first task is technically challenging, e.g. sealing the experiment against ambient pressure withstanding pressure gradients of up to several hundred MPa. The second task requires to combine HP conditions with optical technologies, in the case of biophysical studies of cells and tissues as microscopy techniques. Live cellmicroscopy has been developed to very advanced levels however, HP has rarely been addressed. From such HP experiments, molecular reactions of viable cells that are specific to high pressure stress can be obtainedthat are not available by other biophysical techniques. Our goal is to combine microscopy and engineering expertise to develop a novel HP vessel suitable for multiphoton imaging of living cells under HP. The novel optical chamber will be designed to withstand pressures up to 400 MPa that represent a wide pressure range interesting for eukaryotic and prokaryotic cells. We will implement new optical gradient-index lens technology directly into the pressure vessel rather than using glass windows and external lenses. Then, the system will be applied to investigate (i) Ca2+ transients in field-stimulated mammalian skeletal muscle cells and (ii) resting Ca2+ levels in quiescent cells under various ambient pressure profiles from atmospheric to deep seaenvironments and "pressure X exposure time" products. This will reveal novel biophysical insights into the pressure-induced alterations of excitation-coupled Ca2+ homeostasis in skeletal muscle. Using second-harmonic generation microscopy of myosin, we will determine pressure thresholds and dynamics of sarcomere disintegration for muscle cells. This approach allows to study specific mechanisms for pressure-induced organ damage to skeletal muscle that can explain prime exposure limits for mammals. Theestablished new microscopy technique will provide a novel tool to extend contemporary microscopy to new pressure horizons and to optically study biophysical cell processes for different cell types which will offer newvenues for high pressure biophysics in many organisms of varying complexity.