Figures and data in Contractile force assessment methods for in vitro skeletal muscle tissues

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7 figures and 2 tables

Figures

Figure 1

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Skeletal muscle structure and requirements for contractile force production.

(Top) Summary of the main requirements to enable contractility in skeletal muscle in vitro models. (Middle) Representation of the muscular hierarchy and (bottom) summary of contractile stimuli and contraction profiles.

Figure 2

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Cross-sectional area (CSA) in 2D and 3D muscle models.

(A) Myotube CSA estimated as an elliptical shape from the thickness and the width of the cell. (B) CSA of 3D muscle constructs can be estimated by approximation to different shapes (circle, in left panel), or calculated from immunohistochemical sections. Effective-CSA is known as the area occupied by myotubes (red area in the right panel).

Figure 3

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Cantilever deflection setup.

(A) The beam deflects due to myotube contraction (Left). In this case, cantilever deflection is interrogated by a laser beam and detected using a photodetector (Right). Commonly, cantilever arrays are made of Silicon (Si) or PDMS. Different coatings (FN, laminin, collagen I) have been tested to improve cell attachment and longer culture times. (B) Human myotubes on silicon cantilevers in bright field, top view (top) and immunostained for Myosin Heavy chain, side view (above). Scale bar: 50 µm. (C) Representative images from healthy and DMD myotubes at baseline (i and iii) and peak stress (ii and iv). Blue rectangles represent film length. Red lines represent the tracking of the film edge. Yellow arrows represent the distance between the projected film length and the unstressed film length. The yellow horizontal lines represent the change in projected film length from baseline stress (top bar) to peak stress (bottom bar).

© 2014, Elsevier. Figure 3B is reprinted with permission from Figure 1 from Smith et al., 2014. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

© 2016, Nesmith et al. Figure 3C is reprinted with permission from Figure 5B from Nesmith et al., 2016 (published under a CC BY-NC-SA 3.0 license). It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

Figure 4

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Post Deflection features.

(A) In vitro skeletal muscle is grown between two micropost which serve as anchors (tendons). As muscle contracts in response to a stimulus, posts bend proportionally. By tracking these displacements and knowing the mechanical characteristics of the platform, the force exerted by the muscle can be quantified. (B) Micropost displacement due to miniature bioartificial muscle (mBAM) contraction in response to a maximum tetanic electrical stimulus. Scale bar: 100 µm. (C) Formation of human skeletal muscle micro-tissue (hMMTs). Phase-contrast images depicting the remodeling of the ECM by human myoblast over time. Muscle construct immunostained (2 weeks) for sarcomeric α-actinin (SAA, red) and counterstained with DRAQ5 (1, 5-bis{[2-(di-methylamino)ethyl]amino}–4, 8-dihydroxyanthracene-9, 10-dione) nuclear stain in blue. Scale bar: 500 µm. Reprinted from Figure 2A and C from Afshar et al., 2020.

© 2008, John Wiley and Sons. Figure 4B is reprinted with permission from Figure 4A from Vandenburgh et al., 2008 with permission from John Wiley and Sons. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.

Figure 5

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Force transducers.

(A) In vitro 3D tissue is grown between two anchors. To assess contraction force, one of its sides is connected to a force transducer which will evaluate the force exerted by the muscle due to stimuli. (B) Representative contractile properties of hPSC-derived iSKM bundles. TRiPS-derived bundle (4 weeks) shows increases in contractile force with an increase of stimulation frequency up to the formation of tetanic contraction. Specific force and tetanic-to-twitch ratio of H9 and TRiPS-derived bundle (2 weeks) and (C) (Left) two-week differentiated iSKM bundles pair anchored within a nylon frame. (Right) Representative immunostaining of dense, uniformly distributed myotubes in bundle-CSA. Panel B reprinted from Figures 3A, B and 4A from Rao et al., 2018.

Figure 6

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Overview of the different techniques used to measure contractile force in vitro.

* Represents de % of studies that have performed this measurement.

Figure 6—source data 1 Editable version of Figure 6.: https://cdn.elifesciences.org/articles/77204/elife-77204-fig6-data1-v1.xlsx
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Figure 7

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Functional characteristics of in vitro 2D and 3D skeletal muscle tissues from C2C12 and human sources (immortalized, iPSC and primary myoblast).

(A) Whole cross-sectional area (CSA) of muscle tissues. (B) Tetanic-to-Twitch ratio was calculated from data within the same study, except for bar with a diagonal pattern in post deflection, which was calculated from two different studies. (C) Twitch and Tetanic specific force measure in the three platforms for C2C12 constructs and (D) Human source. Data is presented as mean ± SEM. *p < 0.05, unpaired t-Test.

Tables

Table 1

Maximum contractile force data from in vitro muscle models measured by the three main platforms.

	Cell source	Evaluation time	Size	CSA(mm²)	Twitch contraction		Tetanic contraction		Tetanic-Twitch Ratio*	References
	Cell source	Evaluation time	Size	CSA(mm²)	CF (µN)	sF (kN/m²)	CF (µN)	sF (kN/m²)	Tetanic-Twitch Ratio*	References
Cantilever deflection	C2C12 myoblasts (mouse)	Day 7	50 μm (Width)ⁱ 33 µm (Thickness)*	0.001308*	0.54 ± 0.02	0.41*	1.01 ± 0.14	0.77*	1.87	Fujita et al., 2010
	Rat myoblasts (embryonic)	Day 10–13	22.5 µm (Width)ⁱ 10 µm (Thickness)	0.000176*	0.23*	1.3	__	__	__	Wilson et al., 2010
	C2C12 myoblasts (mouse)	Day 6	12.75 µm (Width)* 8–9 µm (Thickness)	0.0000851*	0.80*	9.4 ± 4.6	__	__	__	Sun et al., 2013
	Rat myoblasts (embryonic)	Day 12–14	11.7–23.4 μm (Width) 7.9–13 μm (Thickness)	0.000144*	0.04–0.26 Stoney’s Eq. 0.03–0.18 FEA	0.359–1.70 Stoney’s Eq. 0.168–1.17 FEA	__	__	__	Pirozzi et al., 2013
	Primary human myoblast	Day 23	10 µm (Width)ⁱ 6.67 µm (Thickness)*	0.000052*	0.14^g	2.69*	__	__	__	Smith et al., 2014
	Rat myoblasts (adult)	Day 4–7	16.74 µm (Width)* 11.16 µm (Thickness)^g	0.000146*	0.17^g	1.15*	__	__	__	McAleer et al., 2014
	Human myoblasts	Day 3–6	12.11 μm (Width)^g 8.07 µm (Thickness)*	0.0000767*	0.78*	9.98^g	__	__	__	Nesmith et al., 2016^#
	Human induced pluripotent stem cell	Day 14	11.82 μm (Width)^g 10.35 μm (Thickness)^g	0.0000961*	0.38*	3.98^g	__	__	__	Badu-Mensah et al., 2020
	Human induced pluripotent stem cell	Day 10–11	9.30 μm (Width)^g 6.2 μm (Thickness)*	0.0000452*	0.12 ± 0.02	2.65*	__	__	__	Guo et al., 2020
	Chick myoblasts	3 weeks	11.24 µm (Width)^g 7.49 μm (Thickness)*	0.0000661*	1.44*	21.89^g	3.31*	50^g	2.28	Santoso et al., 2021
	C2C12 myoblasts (mouse)		16.30 µm (Width)^g 10.86 μm (Thickness)*	0.000139*	0.027	0.2^g	0.018	0.129^g	0.64
	Human myoblasts		14.02 µm (Width)^g 9.34 μm (Thickness)*	0.0001028*	0.020	0.2^g	0.019	0.182^g	0.91
	Human induced pluripotent stem cell	Day 7–10	22.5 μm (Width)* 15 μm (Thickness)	0.000265*	0.26*	0.986^g	0.52	1.986^g	2.01	Al Tanoury et al., 2021
Post deflection	Primary Mouse myoblasts	Day 1–12	2 mmⁱ	3.14*	__	__	42.5^g	13.53*	__	Vandenburgh et al., 2008^#
	C2C12 myoblasts (mouse)	Day 14	0.14 ± 0.01 mm	0.0125 (active) 0.0012 (effective)	1.4*	0.11 (active)* 1.12 (effective)*	__			Sakar et al., 2012
	C2C12 myoblasts (mouse)	Day 6	0.32 mmⁱ	0.079*	__	__	57.5 ± 12.8	0.72*		Shimizu et al., 2017^#
	Primary human myoblasts	Day 11	0.85 mm*	0.566ⁱ	79.44ⁱ	0.14*	428.57ⁱ	0.76*	5.42	Mills et al., 2019
	Derived-Myoblasts from Human Dermal Fibroblast	Day 4–10	0.30 mmⁱ	0.120*	__	__	12.2 ± 5.3	0.10*	__	Shimizu et al., 2020
	Primary human myoblasts	Day 7–14	0.71 mm*	0.395*	__	__	192*	0.49*	__	Afshar et al., 2020
	Immortalized human myoblast	Day 8	0.4 mm	0.125*	__	__	28.5 ± 10.5	0.23	__	Nagashima et al., 2020
	Immortalized human myoblast	Day 7–14	0.47 mm*	0.17 ± 0.03	200 ± 40	1.17	1100 ± 300	6.47	5.52	Hofemeier et al., 2021
	Immortalized human myoblast	Day 10	0.49 mmⁱ	0.189*	118.01^g	0.62*	201.89^g	1.07*	1.72	Ebrahimi et al., 2021
Force tranducers	Rat myoblasts (adult)	Day 31 ± 4	0.49 ± 0.04 mm	0.188*	215 ± 26	1.14*	440 ± 45	2.9 ± 0.5	2.54	Dennis and Kosnik, Ii, 2000
	Rat myoblast (Extensor digitorum longus)	Day 32 ± 4	0.17 mm*	0.024 ± 0.009	162 ± 125	6.75*	281 ± 218	11.70*	1.73	Dennis et al., 2001
	Rat myoblasts	3 weeks	0.18 ± 0.01 mm	0.0246*	329 ± 26.3	13.37*	805.8 ± 55	32.75	2.45	Huang et al., 2005
	Rat myoblasts	Day 16–18	0.25 mm^g	0.048*	102^g	2.12*	212^g	4.41*	2.08	Larkin et al., 2006
	C2C12 myoblasts (mouse)	Day 5–8	0.21 mm*	0.0978ⁱ	71.39*	0.73 ± 2.13	86.06*	0.88 ± 0.48	1.20	Fujita et al., 2009^#
	C2C12 myoblasts (mouse)	Day 2–17	0.2 mm*	0.031*	33.2	1.06	__	__	__	Yamamoto et al., 2011^#
	Rat myoblasts (neonatal)	Day 14	2.7 ± 0.18 mm (Bundle)	5.72 (Bundle)*	1680 ± 320	0.29* 5.5 ± 0.6 (effective)	2840 ± 500	0.50* 9.4 ± 0.7 (effective)	1.72	Hinds et al., 2011
	C2C12 myoblasts (mouse)	Day 7	0.40 mm	0.13*	18.3 ± 2.4	0.15*	34.5 ± 2.8	0.276*	1.84	Sato et al., 2013^#
	Rat myoblasts	2 weeks	1.38 mm (Bundle)* 0.9 mm (F-actin+) *	1.50 (Bundle)^g 0.63 ± 0.05 (F-actin+)	17830 ± 1000	11.89 (Bundle)* 28.30 (F-actin+)*	28800 ± 930	19.2 (Bundle)* 43.39 ± 3.82 (F-actin+)	1.61	Juhas and Bursac, 2014^#
	Primary Human myoblast	4 weeks	2.5 mmⁱ	4.91*	701^g	0.14*	1460^g	0.30*	2.14	Madden et al., 2015
	C2C12 myoblasts (mouse)	Day 14	0.5 ± 0.08 mm	0.19*	81.26^g	0.42*	151.37^g	0.79*	1.88	Ikeda et al., 2016^#
	C2C12 myoblasts (mouse)	3 weeks	0.6 mmⁱ	0.28*	166.3 ± 59.4	0.59*	__	__	__	Nakamura et al., 2017
	hPSC derived human myoblasts	2 weeks	0.42 mmⁱ	0.14*	140^g	1.04	402^g	3.00*	2.88	Rao et al., 2018
	C2C12 myoblasts (mouse)	Day 14	0.98 mmⁱ	0.756*	48.39 ± 3.49	0.06	47.74 ± 0.31	0.06	1	Capel et al., 2019
	Primary Human Myoblast	2 weeks	0.62 mmⁱ	0.30*	1700 ± 130	5.70*	3400 ± 180	11.40*	2	Khodabukus et al., 2019
	hPSC derived human myoblasts	4 weeks	0.28 mmⁱ	0.06*	1393 ± 342	23.21*	2924 ± 517	48.73*	2.09	Xu et al., 2019
	C2C12 myoblasts (mouse)	10 Days	0.99 mm	0.77*	1360 ± 210	1.77*	1930 ± 120	2.50*	1.41	Akiyama et al., 2021
	Primary Human Myoblast	Day 17–19	1.39 mmⁱ	1.51*	__	__	175*	0.13*	__	Alave Reyes-Furrer et al., 2021

*Recalculated data; ^gData extract from a graph; ⁱData extract from an image; ^#Studies with where maximal instantaneous CF data was used.

Table 1—source data 1 Additional information specific to the experimental setup and stimulation parameters.: https://cdn.elifesciences.org/articles/77204/elife-77204-table1-data1-v1.pdf
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Table 2

Summary of several skeletal muscle disease models in post deflection and force transducer platforms.

Key parameters like drugs, change in CF (%∆CF) and other observed effects are detailed.

Disease model	Drug	Platform	Cell source	%∆CF(Dose)	Observed effect	Reference
Atrophy	Dexamethasone	Post deflection	C2C12 myoblast (mouse)	–53% (100 µM)	Increase in the expression of Atrogin-1 (2.6) and MuRF-1 (2.2) Decrease in number of fibers with striation patterns (20% vs 8%)	Shimizu et al., 2017
			Immortalized human myogenic cells	–57% (100 µM)	Increase in the expression of Atrogin-1 and MuRF-1. Expression of FOXO3 and KLF15	Nagashima et al., 2020
			Primary Human myoblast	–85% (10 nM)	Dose-dependent decrease in myotube width	Afshar et al., 2020
			C2C12 myoblast (mouse)	–48% (1 mM)		Yoshioka et al., 2020
		Force transducer	Primary Human myoblast	–67% (25 µM)	Decrease in myotube diameter (25 µM, 12%) Decrease in effective-CSA (60%) Decrease in injury biomarkers CK and LDH	Khodabukus et al., 2020
		Force transducer	C2C12 myoblast (mouse)	–70% (100 µM)	Decrease in myotube-CSA (37%)	Aguilar-Agon et al., 2021
Hypertrophy	IGF-1	Force transducer	Rat myoblast	+ 31% (75 ng)	Increase in CF (75 ng, 31%) Slow Time to peak twitch force (25 ng, 26%)	Huang et al., 2005
		Force transducer	Primary Human myoblast	+ 28% (0.5 mg/ml)	Increase in myotube diameter (0.5 mg/ml, 21%) Increase in injury biomarkers CK and LDH	Khodabukus et al., 2020
		Post deflection	Primary Mouse myoblast	+ 66% (100 ng/ml)	Increase in fiber-CSA (41%)	Vandenburgh et al., 2008
			Immortalized control C57 and mdx myoblast	+ 93% (0.01 µM)		Vandenburgh et al., 2009
			C2C12 myoblast (mouse)	+ 25%	Increase in CF in Dex-induced atrophic tissues (45%*)	Shimizu et al., 2017
			Derived myoblast from human dermal fibroblast	+ 72% (100 ng/ml)	Decrease in CF in non-cryopreserved cells (100 ng/ml, 79%*)	Shimizu et al., 2020
Statin-induced myopathy (Rhabdomyolysis)	Lovastatin	Force transducer	Primary Human myoblast	–75% (2 µM)	Dose-dependent lipid accumulation	Madden et al., 2015
		Force transducer	Primary Human myoblast	–53% (5 µM)	Dose-dependent lipid accumulation	Ananthakumar et al., 2020
		Post deflection	Immortalized human myogenic cells	–75% (2 µM)	Increase in the expression of Atrogin-1 and MuRF-1.	Nagashima et al., 2020
	Cerivastatin	Force transducer	Primary Human myoblast	–50% (50 nM)	Decrease in CF (50 nM, 50%*) Dose-dependent lipid accumulation	Madden et al., 2015
			Primary Human myoblast	–85%	Reduction in myotube diameter Dose-dependent decrease in injury biomarkers CK and LDH	Vandenburgh et al., 1996
			Human Skeletal myoblast	–40% (100 nM)	Decrease in CF Decrease in passive force Myofibers Sarcomere degradation	Zhang et al., 2018
		Post deflection	Primary Human myoblast	–62% (10 nM)	Decrease in CF (10 nM, 62%*) Dose-dependent decrease in myotube width	Alave Reyes-Furrer et al., 2021