regeneration model of 2d and 3d skeletal muscle tissue by...

Regeneration model of 2D and 3D skeletal muscle tissue by treatment with CoQ10Denise M. Almora1,2, Tatsuya Osaki 3,, Roger D. Kamm1,3.

1Department of Biological Engineering, 3Department of Mechanical Engineering. Massachusetts Institute of Technology.2Department of Biomedical Engineering. Florida International University.

Sarcopenia and Myopenia are muscular diseases associated with aging along with theloss of muscle mass and strength, which affects balance and the ability to performtasks of daily living. Microfluidics hold great

promise in aiding the study of these diseases

and the drugs candidates for their treatment.

3D microfluidic models more closely mimic the

behavior of the cells, tissues and organs in vivo

than traditional 2D cultures (1). Many studies

indicate that drugs such as simvastatin affect

the ability of the skeletal muscles to repair and regenerate (2). Skeletal muscle, themost abundant tissue of the body, has the ability to regenerate new muscle fibersafter it has been damaged by injured. Studies show that the Coenzyme Q10 (CoQ10)can assist muscle regeneration (3). The purpose of this study was to create a modelof injured skeletal muscle with the application of 5µM (regular patient dose) or 10µM of simvastatin for 48 hours, and study how the muscle regeneration occurs if thecells are supplied with enough CoQ10. Furthermore, C2C12 ChR2 muscle cells weredifferentiated into myotubes using regular differentiation medium, and usingoptogenetics, with a 450 nm wavelength light source. The effect of the CoQ10 wasexamined using confocal imaging and analysis of gene expression.

Motivation

Acknowledgments

Background

Methods

2D culture muscle degeneration with simvastatin

Expression of F-actin/ alpha-actinin in 2D culture

mRNA Analysis for 2D culture

• Of the elderly U.S. population, approximately 45% is sarcopenic and 20% isfunctionally disabled of muscle tissues.

• The estimated healthcare cost attributable to sarcopenia in the US was $18.5billions, or about 1.5% of total healthcare expenditures.

• Therefore, it is important to seek new effective drug candidates to treat thesemuscle injuries.

• Pharmaceutical companies are striving to develop new methods of drug delivery inour body and screen the drug candidate for sarcopenia and other musculardiseases.

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Gene expression muscle differentiation comparison between optogenetically trained muscle and control sample

• Optogenetic training mimics the activity of muscle contraction, and it shows theexpression of the MHC, MYL-1 and MYL-3 to be upregulated with respect to thecontrol.

• The morphology of the cells after 5µM/10µM of statins changed with respect to thehealthy muscle for both Opt-control and control.

• Changes in the cytoskeleton are observed after the statin treatment. Not observablerecovery in the CoQ10 treatment for 24 hours.

• Visible morphological change observed between the non-treated muscle and thetreated with simvastatin. Decrease in the density of the sarcoplasm, thus the actinfilament is observed in the immunostaining analysis.

• No damage in the sarcomeres is observed in the control sample with CoQ10treatment, meaning that the CoQ10 does not cause damage to healthy muscle.

• All genes were upregulated with CoQ10treatment after 10 µM of simvastatin.

• Higher concentration of statin, decreasedMYL-1, MYL-3, but increased Caspase3 andCaspase9.

• Treatment of CoQ10 after 5 µM ossimvastatin, upregulated every gene exceptfor MHC and MYL-1.

5 µM simvastatin for 48 h 10 µM simvastatin for 48 hNo drug added (Control)

No drug added (Opt-Control) 5 µM simvastatin for 48 h 10 µM simvastatin for 48 h

Optogenetically differentiated 2D culture

α actinin|F-Actin|DAPI

No drug added 5 µM simvastatin for 48 h CoQ10 treatment for5µM simvastatin

CoQ10 treatment for10 µM simvastatin

Control with CoQ10

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MHC: myosin heavy chain, Cas3: Caspase 3, Cas9: Caspase9, MYL-1: Myosin Light Chain 1, MYL-3: Myosin Light Chain 3

10 µM simvastatin for 48 h

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3D Co-Culture HUVECS/ Myotubes

The microfluidic device is composed of two main parallel channels. Each channels are 500-600μm in a diameter and 12 mm in length. In one channel C2C12 mouse differentiated myoblastscells are seeded. After 7 days of differentiation to myotubes in the channel, simvastatin is addedfor 48 hours. Then, the endothelial cells are be seeded in the second parallel channel and treatedwith 10µM of CoQ10 for 24 hours .

ConclusionsSimvastatin drug caused damage to the muscle physiologically. Moreover, the control with CoQ10did not show morphological difference compared to the control with no drug added, meaningthat the CoQ10 does not affect muscle health. However, the time used for treatment with CoQ10(24 h) was not enough for quantification of muscle rebuilding. Another study with a higherconcentration of CoQ10 (20 μM) could serve to analyze if higher concentrations of the drug aidfaster muscle regeneration.

• Performing DNA Analysis of 3D model of injured and treated muscle.• Re-doing the experiment using a higher concentration of CoQ10 and/or more time for the

drug to act. Also, determining other candidate drugs to study muscle force recovery.

Future Directions

MyoD|F-actin|DAPI

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Healthy vs Sarcopenic muscle

References: 1. Haase,K.; Kamm, R.D. (2017). Journal or Regenerative Medicine. 2. Krupa, D. (2017) TheAmerican Physiological Society. 3. Littarru, G.P., Langsjoen, P. (2010). The Ochsner Journal.

C2C12 muscle cells differentiation

F-actin/ α actininConfocal Imaging

Addition of 5/10 µM simvastatin for 48 hours

Treatment with CoQ10 for 24 hours

Co-Culture with HUVECS



C2C12 cells differentiation with optogenetic training

* with respect to control** with respect to simvastatin treated models

Myotubes (left) / HUVECs (right)

regeneration model of 2d and 3d skeletal muscle tissue by...

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