Calcium‐dependent deceleration of the cell cycle in muscle cells by simulated microgravity

Abstract

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.

TRPC6 in simulated microgravity of intervertebral disc cells

“…For the experimental phase, cells were cultured in reduced-serum DMEM/F12 (5% FCS) without antibiotics to avoid any possible interaction of aminoglycoside antibiotics with TRP channel activity [28].…”

“…IVD cells (n = 4-5, seeded at 6000 cells/cm 2 in T25 flasks (150,000 cells per flask)) were cultured for 24 h prior to simulated microgravity. Flasks were completely filled with culture medium to avoid air bubbles and closed airtight with a filter-less cap [28]. Flasks were mounted onto the RPM (integrated miniaturized CO 2 incubator), whereas corresponding control samples (1 g) were cultured in the same conditions and simultaneously kept in a standard CO 2 incubator [28].…”

“…Flasks were completely filled with culture medium to avoid air bubbles and closed airtight with a filter-less cap [28]. Flasks were mounted onto the RPM (integrated miniaturized CO 2 incubator), whereas corresponding control samples (1 g) were cultured in the same conditions and simultaneously kept in a standard CO 2 incubator [28]. Treated cells were analyzed for proliferation (up to 5 days of treatment), cell cycle progression (24 h of treatment), cellular senescence (72 h of treatment) and TRP channel expression (72 h of treatment) and compared to untreated controls.…”

“…Aside from TRPC6, the isoform TRPC1 may also be of specific relevance. TRPC1 and TRPC6 are both known to affect cell cycle progression in various cell types [27][28][29], and TRPC6 was shown to modulate cellular senescence [30]. Therefore, their modulation and (dys-)regulation may play a crucial role in IVD health, but also degeneration.…”

“…TRP channels are therefore interesting candidates when aiming to link IVD mechanobiology with IVD ageing and degeneration. Interestingly, simulated microgravity was shown to depress TRPC1 expression associated with an accumulation of cells into G2/M, effectively stalling proliferation of mouse myoblasts [28,31].…”