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

• Applicant: Professor Oliver Friedrich
• Subject Area: Biophysics ; Medical Physics, Biomedical Technology ; Cell Biology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 255191772

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.

• DFG Programme: Research Grants
• International Connection: Australia, United Kingdom
• Participating Persons: Professorin Dr. Rosalind Allen; Professor Boris Martinac, Ph.D.