Development of a functional sarcopenia model utilizing a microcantilever microphysiological system as a phenotypic disease model

Figures

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  Development of the first defined in vitro functional model of sarcopenia on a microcantilever platform. This study recapitulates physiological signatures of disease progression characterized by loss of muscle mass and function and an increase in reactive oxygen species

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  Fig. 1. X-ray photoelectron spectroscopy (XPS) analysis showing incorporation of the extracellular matrix proteins elastin and collagen on the DETA surfaces. a XPS spectrum of coverslips modified with the DETA-silane. b XPS spectrum of the protein coating (elastin and collagen) on DETA-silane modified coverslips. c Si2p high-resolution XPS spectrum. d C1s high-resolution XPS spectrum indicated the growth of a carbonyl carbon peak at 288 eV as well

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  Fig. 2. Sarcopenia induction using TNFα: a timeline indicating the culture conditions employed for the control and sarcopenic phenotype. For both conditions, myoblasts were allowed to proliferate in myocult medium, then primed in DKHI medium, followed by switching to differentiation medium; TNF-alpha (3 nM) was added only to the sarcopenic system. b Phase image for control condition at day − 2. c Phase image for control conditions at day 0. d Phase image for control condition at day 4. e Phase image for sarcopenic condition day − 2. f Phase image for sarcopenic condition day 0. g Phase image for sarcopenic condition day 4

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  Fig. 3. Decrease in myotube width upon sarcopenia induction. Representative phase images in the control condition, a day 10, b day 14, c day 17, d day 21, e day 28, and f day 32. Representative phase images in the sarcopenic condition, g day 10, h day 14, i day 17, j day 21, k day 28, and l day 32. Scale bar equals 50 µm. Bottom graph showing quantification of myotube width over 32 days in control and sarcopenic conditions. This data was sampled from three independent experiments. Error bars represent standard error

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  Fig. 4. Immunocytochemical analysis of SKM myotubes with myosin heavy chain protein (red) with DAPI labeled nuclei (blue). Representative images from control group. a Image showing myosin heavy chain and nuclear stain. b Phase image. c Nuclear stain using DAPI: representative images from sarcopenic condition. d Merged image showing myosin heavy chain and nuclear stain. e Phase image indicating myotubes. f DAPI stain. Arrows indicate striated myotubes. Scale bar = 50 µm

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  Fig. 5. Quantification of contractile amplitude for control (blue) and sarcopenia (orange) induced SKM myotubes. Representative myotube contractions on day 14 for cultures stimulated at two frequencies: a 0.33 Hz and b 0.5 Hz. Representative myotube contraction profile on day 32 at stimulation frequencies of c 0.33 Hz and d 0.5 Hz. Amplitudes plotted for control and sarcopenic conditions on days 14, 17, 21, 28, and 32, at stimulation frequencies of e 0.33 Hz and f 0.5 Hz. The amplitudes for the sarcopenic condition were normalized to the control group on corresponding days

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  Fig. 6. Measurement of fatigue index by stimulation of cultures at 1 Hz, 2 Hz, and 4 Hz. Representative myotube contraction traces on days 14, 17, 21, 28, and 32 at stimulation frequencies of 1 Hz, 2 Hz, and 4 Hz for control (blue) and sarcopenic (orange) conditions. Corresponding quantification of fatigue indices at three different frequencies is shown in the lower panel

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  Fig. 7. SKM force measurements with the microcantilever system. Representative force characteristics for control and sarcopenic conditions on a day 14, b day 17, c day 21, and d day 28, upon repeat testing. e Phase images under bright field showing SKM myotubes on the microcantilever chip on day 14 in control conditions. f Phase image under bright field showing the presence of myotubes on day 14 in sarcopenic conditions. g Average amplitude of photodetector voltage representing force produced by SKM myotubes in control and sarcopenic conditions on days 14, 17, 21, and 28

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  Fig. 8. Detection of ROS in sarcopenic versus control cells. a Flow cytometry analysis revealed that sarcopenic cells at day 4 exhibited higher ROS than control cells. Gating strategy for single cells, followed by FSC-H vs SSC-H to isolate the myoblast population. Viability was further confirmed by DRAQ5 staining of gated singlets (data not shown). b DCF labeling of cells at day 4 was detected in the FITC channel. c Quantification of DCF-positive myoblasts as a readout for ROS based on day 4 flow cytometry. d DCF labeling of cells at day 40. e Quantification of mean fluorescence intensity (MFI) based on gated DCF-positive populations. f Confocal images showed DCF labeling of control and sarcopenic cells, further processed using ImageJ

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  Fig. 9. Viability and mitochondrial potential measurements in sarcopenic versus control cells. a Myoblasts cultured under control and increasing concentrations of TNFα for 4 days were labeled with TMRE, followed by fluorescence measurements at emission (488 nm)/excitation (575 nm) using a microplate reader. b In parallel, viability was acquired by incubating the same batch of cells with Cell Titer Glo reagent for 10 min, followed by luminescence recordings using a microplate reader. c Control and sarcopenic cells were labeled with TMRE and fluorescence measurements were acquired on day 8 as previously described

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  Fig. 10. Schematic indicating the development of sarcopenic conditions. Muscle precursor cells differentiate into myoblasts, in which healthy cells undergo primary and secondary fusion forming thick and contractile multi-nucleated myotubes. In contrast, TNFα acts as a mitogen on myoblasts, inducing their proliferation and causing a decrease in their fusion rate into myotubes. The aging process and the accumulation of ROS lower the functional contractile force of sarcopenic cells