No abstract

The effect of differentiation on the transverse mechanical properties of mammalian myocytes was determined by using atomic force microscopy. The apparent elastic modulus increased from 11.5 +/- 1.3 kPa for undifferentiated myoblasts to 45.3 +/- 4.0 kPa after 8 days of differentiation (P < 0.05). The relative contribution of viscosity, as determined from the normalized hysteresis area, ranged from 0.13 +/- 0.02 to 0.21 +/- 0.03 and did not change throughout differentiation. Myosin expression correlated with the apparent elastic modulus, but neither myosin nor beta-tubulin were associated with hysteresis. Microtubules did not affect mechanical properties because treatment with colchicine did not alter the apparent elastic modulus or hysteresis. Treatment with cytochalasin D or 2,3-butanedione 2-monoxime led to a significant reduction in the apparent elastic modulus but no change in hysteresis. In summary, skeletal muscle cells exhibited viscoelastic behavior that changed during differentiation, yielding an increase in the transverse elastic modulus. Major contributors to changes in the transverse elastic modulus during differentiation were actin and myosin.

Effects of mechanical forces exerted on mammalian skeletal muscle cells during development were studied using an in vitro model to unidirectionally stretch cultured C2C12 cells grown on silastic membrane. Previous models to date have not studied these responses of the mammalian system specifically. The silastic membrane upon which these cells were grown exhibited linear strain behavior over the range of 3.6-14.6% strain, with a Poisson's ratio of approximately 0.5. To mimic murine in utero long bone growth, cell substrates were stretched at an average strain rate of 2.36%/day for 4 days or 1.77%/day for 6 days with an overall membrane strain of 9.5% and 10.6%, respectively. Both control and stretched fibers stained positively for the contractile protein, alpha-actinin, demonstrating muscle fiber development. An effect of stretch on orientation and length of myofibers was observed. At both strain rates, stretched fibers aligned at a smaller angle relative to the direction of stretch and were significantly longer compared to randomly oriented control fibers. There was no effect of duration of stretch on orientation or length, suggesting the cellular responses are independent of strain rate for the range tested. These results demonstrate that, under conditions simulating mammalian long bone growth, cultured myocytes respond to mechanical forces by lengthening and orienting along the direction of stretch.

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C2C12 murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of myoblasts cultured on microcarrier beads in teflon bags, myoblasts cultured in the bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and alpha-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p < 0.01) and tropomyosin (63% decrease, p < 0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentiation. These results may have implications for muscle growth and repair during spaceflight.