Titin (also known as connectin) is a giant protein with a wide range of cellular functions, including providing muscle cells with elasticity. Its physiological extension is largely derived from the PEVK segment, rich in proline (P), glutamate (E), valine (V), and lysine (K) residues. We studied recombinant PEVK molecules containing the two conserved elements: ≈28-residue PEVK repeats and E-rich motifs. Single molecule experiments revealed that calcium-induced conformational changes reduce the bending rigidity of the PEVK fragments, and site-directed mutagenesis identified four glutamate residues in the E-rich motif that was studied (exon 129), as critical for this process. Experiments with muscle fibers showed that titin-based tension is calcium responsive. We propose that the PEVK segment contains E-rich motifs that render titin a calcium-dependent molecular spring that adapts to the physiological state of the cell.

Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C₂C₁₂myotubes by manipulating intracellular Ca²⁺transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser²³¹after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from “fast type” to “slow type” was observed in the C₂C₁₂myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C₂C₁₂myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise.

Electrical impulses are necessary for proper in vivo skeletal muscle development. To fabricate functional skeletal muscle tissues in vitro, recapitulation of the in vivo niche, including physical stimuli, is crucial. Here, we report a technique to engineer skeletal muscle tissues in vitro by electrical pulse stimulation (EPS). Electrically excitable tissue-engineered skeletal muscle constructs were stimulated with continuous electrical pulses of 0.3 V/mm amplitude, 4 ms width, and 1 Hz frequency, resulting in a 4.5-fold increase in force at day 14. In myogenic differentiation culture, the percentage of peak twitch force (%Pt) was determined as the load on the tissue constructs during the artificial exercise induced by continuous EPS. We optimized the stimulation protocol, wherein the tissues were first subjected to 24.5%Pt, which was increased to 50–60%Pt as the tissues developed. This technique may be a useful approach to fabricate tissue-engineered functional skeletal muscle constructs.

System level understanding of the cell requires detailed description of the cell state, which is often characterized by the expression levels of proteins. However, understanding the cell state requires comprehensive information of the cell, which is usually obtained from a large number of cells and their disruption. In this study, we used Raman spectroscopy, which can report changes in the cell state without introducing any label, as a non-invasive method with single cell capability. Significant differences in Raman spectra were observed at the levels of both the cytosol and nucleus in different cell-lines from mouse, indicating that Raman spectra reflect differences in the cell state. Difference in cell state was observed before and after the induction of differentiation in neuroblastoma and adipocytes, showing that Raman spectra can detect subtle changes in the cell state. Cell state transitions during embryonic stem cell (ESC) differentiation were visualized when Raman spectroscopy was coupled with principal component analysis (PCA), which showed gradual transition in the cell states during differentiation. Detailed analysis showed that the diversity between cells are large in undifferentiated ESC and in mesenchymal stem cells compared with terminally differentiated cells, implying that the cell state in stem cells stochastically fluctuates during the self-renewal process. The present study strongly indicates that Raman spectral morphology, in combination with PCA, can be used to establish cells' fingerprints, which can be useful for distinguishing and identifying different cellular states.

To be able to predict antibiotic resistance in bacteria from fast label-free microscopic observations would benefit a broad range of applications in the biological and biomedical fields. Here, we demonstrate the utility of label-free Raman spectroscopy in monitoring the type of resistance and the mode of action of acquired resistance in a bacterial population of Escherichia coli, in the absence of antibiotics. Our findings are reproducible. Moreover, we identified spectral regions that best predicted the modes of action and explored whether the Raman signatures could be linked to the genetic basis of acquired resistance. Spectral peak intensities significantly correlated (False Discovery Rate, p < 0.05) with the gene expression of some genes contributing to antibiotic resistance genes. These results suggest that the acquisition of antibiotic resistance leads to broad metabolic effects reflected through Raman spectral signatures and gene expression changes, hinting at a possible relation between these two layers of complementary information.