Eccentric exercise leads to focal disruptions in the myofibrils, referred to as “lesions”. These structures are thought to contribute to the post-exercise muscle weakness, and to represent areas of mechanical damage and/or remodelling. Lesions have been investigated in human biopsies and animal samples after exercise. However, this approach does not examine the mechanisms behind lesion formation, or their behaviour during contraction. To circumvent this, we used electrical pulse stimulation (EPS) to simulate exercise in C2C12 myotubes, combined with live microscopy. EPS application led to the formation of sarcomeric lesions in the myotubes, resembling those seen in exercised mice, increasing in number with the time of application or stimulation intensity. Furthermore, transfection with an EGFP-tagged version of the lesion and Z-disc marker filamin-C allowed us to observe the formation of lesions using live cell imaging. Finally, using the same technique we studied the behaviour of these structures during contraction, and observed them to be passively stretching. This passive behaviour supports the hypothesis that lesions contribute to the post-exercise muscle weakness, protecting against further damage. We conclude that EPS can be reliably used as a model for the induction and study of sarcomeric lesions in myotubes in vitro.

The identification of large numbers of phosphopeptides from complex samples largely relies on sample fractionation to reduce complexity and allow using large amounts of starting material. For such experiments, commonly fractionation of whole cell lysate digests followed by enrichment of phosphopeptides from the single fractions is performed. We evaluated the tip-based fractionation of batch-enriched phosphopeptides as an alternative method. We compared three tip-based fractionation methods employing strong cation exchange (SCX), strong anion exchange (SAX), and C18 material for basic reversed-phase (BRP) fractionation using HeLa whole cell lysate digests. We show that SCX tips are superior to BRP and SAX tips due to a more efficient retention and distribution of phosphopeptides as well as a better resolution. Furthermore, we show that tip-based fractionation results in a similar performance as fractionation followed by phosphopeptide enrichment of the single fractions and outperforms analysis of unfractionated phosphopeptide-enriched samples with long chromatography gradients. Our fractionation approach using SCX tips is straightforward, reproducible, and requires a fraction of time, effort, and instrumentation compared to those of the fractionation of whole cell lysate digests with subsequent enrichment of phosphopeptides from the single fractions.