Quantitative method for assessing contractile activity of myotubes

BACKGROUND: Myotubes are multinucleated terminally differentiated cells widely used to study changes induced by muscle contractile activity, metabolic disorders, and myopathies. The ability to contract is a key indicator of muscle cell maturity; therefore, quantitative assessment of this property is essential for evaluating the degree of myotube differentiation. Existing methods for assessing evoked contractile activity of myotubes have several limitations, including short analyzed recording intervals and the inability to distinguish true cellular activity from artifacts.

AIM: This study aimed to develop a method for quantitative evaluation of evoked myotube contractile activity that accounts for contraction–relaxation cycles and artifact effects and enables analysis of dozens of contraction–relaxation events.

METHODS: C2C12 myotubes on days 9–11 of differentiation were electrically stimulated for 1 hour using sequential bipolar rectangular pulses with 2 ms duration (45 Hz, 1.7 mA per well) for 300 ms, followed by 700 ms of rest. Contractile activity was recorded on video (40 s at the beginning and end of each session) and quantified using existing methods based on the assessment of displacement within an image region or pixel intensity variation, as well as a newly developed method based on calculating the mean standard deviation of pixel intensity within a moving window.

RESULTS: The proposed method reduced artifact influence (focus drift and particle movement in the medium) on the myotube contractile activity index by an order of magnitude compared with existing approaches, and decreased the coefficient of variation between technical replicates twofold. Extending the analyzed recording duration (to 40 seconds) further reduced variability (by 1.4–2.3 times) compared with shorter video recordings. The Python implementation of the method is available in open access (https://github.com/maxpauel/movindex).

CONCLUSION: This study proposes a quantitative method for evaluating evoked myotube contractile activity, which enables effective elimination of artifacts associated with particle motion in the culture medium and focus instability, as well as assessment of the mean contractile activity within the imaging frame (field of view) over an extended period of time.