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

压力下的骨骼肌功能利用新型高压多光子显微镜技术阐明肌肉 Ca2 信号传导故障和超微结构崩解的机制

• 批准号: 255191772
• 负责人: Professor Dr. Oliver Friedrich
• 金额: --
• 依托单位: The Victor Chang Cardiac Research Institute
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/255191772?language=en
• 关键词: Skeletal; Muscle; Function; under; Pressure; Skeletal; Muscle; Function; under; Pressure

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.

高压（HP）几乎影响所有生物过程。长时间的高静水压力暴露是我们大多数地球生物圈的规则而不是例外。了解HPS对活细胞或整个生物的影响需要控制压力条件，并具有观察诱导的变化的能力，以了解生物如何抵消或适应增加压力环境。第一个任务在技术上具有挑战性，例如将实验固定在环境压力上，但要承受高达几百MPa的压力梯度。第二任务需要将HP条件与光学技术相结合，而将细胞和组织的生物物理研究作为显微镜技术。实时细胞显微镜已开发到非常高级的水平，但是HP很少得到解决。从这样的HP实验中，可以获得特定于高压应力的活细胞的分子反应，可以在其他生物物理技术中获得。我们的目标是将显微镜和工程专业知识相结合，以开发出适合于HP下活细胞多光子成像的新型HP容器。新型的光学室将被设计为承受高达400 MPa的压力，代表着对真核和原核细胞的宽压力范围。我们将直接将新的光学梯度指数镜头技术实施到压力容器中，而不是使用玻璃窗和外部镜头。然后，该系统将用于研究（I）在野外刺激的哺乳动物骨骼肌细胞中的Ca2+瞬变，以及（ii）在从大气到深海环境的各种环境压力谱和“压力X暴露时间”产品的各种环境压力曲线下，静态细胞中的静息Ca2+水平。这将揭示出对骨骼肌中激发偶联Ca2+稳态的压力诱导改变的新生物物理见解。使用肌球蛋白的第二次谐波生成显微镜，我们将确定肌肉细胞的肌节截止的压力阈值和动力学。这种方法允许研究针对骨骼肌造成压力诱导器官损害的特定机制，这些机制可以解释哺乳动物的主要暴露极限。良好的新显微镜技术将为新的压力层延伸到新的压力范围，并为不同的细胞类型的生物物理细胞过程提供新颖的工具，这将为许多不同复杂性的生物体提供高压生物物理学的新颖性。