Simulated Microgravity Conditions Modulate Ca2+ Transport through TRPV4 Channels

Astronauts in space tend to lose ten times as much bone mass each month as severe osteoporosis patients on Earth. Multiple reports on unloading-induced osteoporosis in bed-ridden patients indicate that bone mass correlates positively with mechanical stress. The Transient Receptor Potential Vanilloid Type 4 (TRPV4) is a Ca2+-permeable cation channel which responds to mechanical and osmotic signals in multiple musculoskeletal tissues. This channel links mechanically triggered molecular signaling and Ca2+ levels as contributors of the same molecular pathway leading to increased osteoclast differentiation. In this line of investigation, we simulated microgravity conditions to detect modulation of Ca2+ transport from internal stores through TRPV4 channels using yeast luminescent reporters. The cell culture was introduced to capacity into a cylindrical High Aspect to Ratio Vessel (HARV) in a horizontal (1g, control) or vertical (simulated micro-gravity) position in the Rotary Cell Culture System. The experimental approach required design and implementation of custom optical equipment, which proved successful in determining minute changes in Ca2+ transport by either luminescence or fluorescence measurements. The increase of cytosolic Ca2+ upon release from internal stores was assessed using the luminescence signal generated by the aequorin-coelenterazine system in the presence of Ca2+. Fluorescence measurements of Ca2+ release employed the cell-membrane permeant Ca2+ indicator Fluo-4 AM. The set of experimental data provided in this report demonstrates directly that simulated microgravity conditions induce measurable changes of Ca2+ transport through TRPV4 channels. This modulation of Ca2+ transport was similar to results from hypo-osmotic stress conditions previously described by several studies on the same cells. A better molecular understanding of the contribution of reduced mechanical loading to the decline in bone mass and quality during extended space flight missions and Earth-based bed-ridden conditions is crucial for mitigating pathologies which manifest as severe bone loss.