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Of all our mechanosensitive tissues, skeletal muscle is the most developmentally responsive to physical activity. Conversely, restricted mobility due to injury or disease results in muscle atrophy. Gravitational force is another form of mechanical input with profound developmental consequences. The mechanical unloading resulting from the reduced gravitational force experienced during spaceflight results in oxidative muscle loss. We examined the early stages of myogenesis under conditions of simulated microgravity (SM). C2C12 mouse myoblasts in SM proliferated more slowly (2.23× less) as a result of their being retained longer within the G2/M phase of the cell cycle (2.10× more) relative to control myoblasts at terrestrial gravity. Blocking calcium entry via TRP channels with SKF-96365 (10-20 μM) accumulated myoblasts within the G2/M phase of the cell cycle and retarded their proliferation. On the genetic level, SM resulted in the reduced expression of TRPC1 and IGF-1 isoforms, transcriptional events regulated by calcium downstream of mechanical input. A decrease in TRPC1-mediated calcium entry thus appears to be a pivotal event in the muscle atrophy brought on by gravitational mechanical unloading. Hence, relieving the constant force of gravity on cells might prove one valid experimental approach to expose the underlying mechanisms modulating mechanically regulated developmental programs.

When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedance-based flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation. © 2014 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]