A Window to Cell Signaling under Pressure - Stability of Muscle Structure and Function at High Hydrostatic Pressures applying PiezoGRIN optical Technology

• Applicant: Professor Oliver Friedrich
• Subject Area: Biophysics ; Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 556799444

High Hydrostatic Pressures (HHP) affect virtually all biological processes but are much less well studied than, e.g., fundamental temperature effects. This is due to limited availability of sophisticated pressure-tight metrologies that are required to safely seal off sample volumes against the HHP environment but still faithfully allow sensor data to be recorded from within. In a previous DFG project we developed a new optical pressure vessel, PiezoGRIN that, unlike previous attempts in literature, includes refractive components within the chamber hull design to combine optical and sealing material properties (so-called Poulter-seal). With this system, we successfully extended high-resolution multiphoton microscopy to the HHP realm and studied optical behavior of fluorescent Ca2+-imaging dyes and label-free Second Harmonic Generation (SHG) cellular ultra-structure in pressurized single mouse muscle fibres. As skeletal muscle is the main locomotory organ to enter and escape HHP zones in deep sea animals, fundamental HHP effects on cellular fast Ca2+-signaling and contractility are of huge interest for Marine Biotechnology, Comparative Biology across species and High Pressure Biosciences. In the proposed follow-up project, we intend to now apply the PiezoGRIN-multiphoton approach to perform detailed studies of single muscle cellular (i) excitability, Ca2+-transient and contractility kinetics and (ii) sarcomere actomyosin lattice stability in 3D under high pressures up to 200 MPa in single fibres from three mammalian muscles (mouse edl, soleus, diaphragm DIA) alongside with their myosin heavy chain fibre types assessment following HHP experiments. We hypothesize HHP to raise intracellular Ca2+ levels to impair and slow-down field-stimulation-induced contractility and transients under pressure, and this to a higher extent in slow-twitch fibres (i.e. soleus, DIA). Also, 3D cytoarchitecture we assume to be more pressure resistant in fast-twitch fibres (i.e., edl). The engineering part of the project will upgrade our PiezoGRIN vessel to include electrical field stimulation under HHP employing tailored electroconductive polymer integration, while the biology/biophysics part will address mechanisms of HHP-induced (applying slow-/fast-compression ramps; exposures at given holding HHP) alterations in cellular Ca2+ and contractility kinetics by (a) deriving physico-chemical molecular activation volumes for those kinetics processes, and (b) whether and how the organic osmolyte trimethylamine-oxide (TMAO), highly enriched in deep sea fish species, can serve as an acute piezo-protectant to mammalian muscle cells, in contrast to urea (predominant in terrestrial organisms) that we speculate to exacerbate HHP effects on muscle structure/function. Our project will add substantial fundamental knowledge to basic HHP effects on structure-function relationships in excitable muscle tissue and reveal principal pressure exposure limits for cellular survival.

• DFG Programme: Research Grants