This project was funded by AFM-Telethon and A Foundation Building Strength for Nemaline Myopathies. The authors wish to acknowledge the creator of the products mentioned in this article, IONOptix Inc.

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+Myosin-II+Inhibitor+(N-benzyl-p-toluene+Sulphonamide,+BTS)+on+Contractile+Characteristics+of+Intact+Fast-twitch+Mammalian+Muscle+Fibres.">Effects of a Myosin-II Inhibitor (N-benzyl-p-toluene Sulphonamide, BTS) on Contractile Characteristics of Intact Fast-twitch Mammalian Muscle Fibres.</a> Journal of Muscle Research and Cell Motililty. 26, 135-141 (2005).</li><li>Stehle, R., Kr&#252;ger, M., Pfitzer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Force+kinetics+and+individual+sarcomere+dynamics+in+cardiac+myofibrils+after+rapid+Ca(2+)+changes.">Force kinetics and individual sarcomere dynamics in cardiac myofibrils after rapid Ca(2+) changes.</a> Biophysics Journal. 83, 2152-2161 (2002).</li><li>Najafi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-diastolic+force+pre-activates+cardiomyocytes+and+determines+contractile+force:+role+of+titin+and+calcium.">End-diastolic force pre-activates cardiomyocytes and determines contractile force: role of titin and calcium.</a> Journal of Physiology. 597, 4521-4531 (2019).</li></ol>

Michiel Helmes is shareholder and co-owner of IONOptix Inc.

Described is a protocol to assess the contractile function of myofibrils isolated from human or animal skeletal muscle tissues. The force resolution of this setup has been described before by Chavan et al.12. In short, it is determined by the random fluctuations of the length of the Fabry-P&#233;rot cavity formed between the detection fiber and the cantilever, which produce the dominant part of the noise at the output of the readout (expressed in V) that, multiplied by the deflection sensitivity (expressed in m/V) and by the spring constant of the cantilever (expressed in N/m), provides the force noise. For our setup, the root mean square (rms) noise in air at the output of the readout, sampled at a 1,000 data points/s (sample/s), is approximately 2 mV. For a typical myofibril measurement, a ferrule-top probe is used with a spring constant of ~0.7 N/m (deflection sensitivity &#8764;300 nm/V). This rms value corresponds to a cantilever deflection resolution of 0.6 nm, which translates to a force sensitivity of ~0.37 nN. The force probe is calibrated by pushing the tip of the mounting needle against a weighing scale while keeping the bending of the cantilever equal to a multiple of the wavelength of the readout laser13. This method of calibration entails both the cantilever and mounting needle stiffness as well as possible variations in torque of the cantilever and mounting needle due to speed and magnitude of myofibril contraction. Currently, a setup for assessing myofibril contractility is available, which is based on the detection of a laser deflected from the cantilever, i.e., optical beam deflection (1,700 A; ~1 nN force resolution). This system was developed by Labuda et al. using an optical periscope to guide a laser light towards and away from the cantilever in constraining configurations11. In this system, a myofibril is mounted between the atomic force cantilever and a rigid glass needle. An advantage of the system described here is the higher force sensitivity and signal-to-noise ratio. Furthermore, in this setup, relatively stiff cantilevers can be used, which results in small cantilever deflection when myofibrillar force is applied. This is important, as it allows for force measurements at nearly constant sarcomere length. Finally, compared to the system described by Labuda et al., the system here utilizes similar or identical methods to control the temperature, to induce length changes on the myofibril, and to change the perfusion solutions using a &#415;-glass and fast-step motor. The advantage of the system described by Labuda et al. is that a change of solution composition (between cantilever and optical periscope) does not affect the signal output. In the system described here, the solution composition between cantilever and optical fiber must remain constant. The solution to this limitation is described in more detail below.
Optimization 
The optical force probe in combination with the fast-step perfusion system led to complications. The difference in optical properties between low and high concentration Ca2+ solutions interferes with the force measurements. To prevent backflow of the high calcium solution, a custom flow chamber was engineered (Figure 3). A constant background flow of calcium-free solution is induced from right to left to keep the solution constant between the top of the optical fiber and the cantilever (Figure 3D).
To control the temperature, a Peltier element with liquid cooling is mounted on the flow chamber. This flow chamber is thermally uncoupled from the microscope by mounting it on a plastic adapter. With the Peltier element, controlled by a TEC system, it is possible to control the temperature of the solution over time with 0.1 &#176;C precision. Temperature is monitored by a temperature sensor mounted on the flow chamber. Temperature stability is important due to the nature of the force transducer. The cantilever consists of a gold-coated glass strip, effectively making it a thermometer. Thus, the cantilever bends with temperature changes.
The setup uses a fast-step perfusion system (see Table of Materials) to control the movement of the &#415;-glass. This system allows for perfusion switches within 10 ms. Combining the method of temperature control and solution switching makes this system particularly suitable to measure the kinetics of sarcomere contractility (i.e., rates of force development, tension redevelopment, and relaxation) in myofibrils.
Initially, the downside of using interferometry was the small usable range due to the necessity to use the linear part of the interference curve (&#955;/8, with &#955; being the wavelength of the laser). However, recent innovations eliminated this need by combining wavelength modulation with a lock-in amplifier. Therefore, the system is not limited to a single linear part of the interference curve. This enables the measurement of infinite deflection of the cantilever14. Thus, the range of cantilever deflection readout of this system is greatly enlarged compared to traditional interferometry. Additionally, the force probes described are easy to replace and there are many cantilevers available, with stiffnesses ranging from 0.5 N/m to &#62;20 N/m. Therefore, it is possible to quickly change between cantilevers and select the stiffness most suitable for the experiment conducted.
Challenges 
The current system is a prototype based on a cardiomyocyte measuring system (see Table of Materials). Several components can be improved to provide a better user experience and data of higher quality. First, due to add-ons to the system, vibration and resonance can be an issue that will add noise to the signal. Also, the &#415;-glass holder and fast-step motor attachment method could be improved to make it less prone to vibration.
Second, it is desirable to replace the fast-step motor with a piezo length actuator to increase the speed of solution switching and to obtain a more consistent motion.
Third, the calcium solutions we previously used to activate single striated muscle fibers included propionic acid, but these solutions absorb near-infrared light, interfering with the force measurements. Calcium chloride was used to eliminate the need for propionic acid, which greatly reduced this effect. This issue is inherent to a system based on interferometry and not present when utilizing optical beam deflection.
Fourth, a custom flow bath was engineered to create a laminar flow, to match the flow of the &#415;-glass. This prevents backflow due to turbulence of the calcium-rich solution. Therefore, the solution between the tip of the optical fiber and the cantilever remains constant. The coverslip with the myofibrils can move freely under the flow chamber and therefore, selection of suitable myofibrils is not confined to the small area of the flow chamber.
Reproducibility and variability 
There are several elements of the system and protocol that are important for the degree of reproducibility and variability of the data obtained.
First, the quality of the measurements strongly depends on the quality of the myofibril isolation. Identical protocols yield different qualities and quantities of myofibrils from different biopsies. In some cases, biopsies barely yield usable myofibrils or none at all. Common consensus is that damaged myofibrils will break during contraction and thus are not accounted for in the results.
Second, there is uncertainty in the determination of the cross-sectional area of the myofibril. Due to technical constraints, it is possible to measure the width of the myofibril in only one plane. Therefore, to calculate the cross-sectional area we assume that the width and depth are equal. When force is normalized to a cross-sectional area to calculate maximal active tension, one should be aware of this assumption.
Mounting of myofibrils due to myofibril mounting angle, position, and the integrity of the glue. 
Although mounting angle and position can largely be controlled visually, small variations between myofibrils might be present. Glue integrity has not been investigated extensively. However, glue integrity can be verified by monitoring the sarcomere length in the myofibril before and after activation. When more sarcomeres are between the glue after a protocol, this suggests that slippage of the myofibril in the glue has occurred. Consequently, this myofibril should be excluded from the dataset.
Other applications of the setup: Calcium preactivation in cardiomyocytes isolated from rat left ventricle 
In addition to assessing the contractile function of myofibrils, the system can also be used to measure cardiomyocyte mechanics. For example, Figure 11 illustrates the use of membrane-permeabilized single cardiomyocytes isolated from rat left ventricle21. Contrary to the experiments described above, relaxing solution was changed and activating solution was kept constant. Each cardiomyocyte underwent five sets of activations, exposing it to a 2 &#181;M free calcium solution for 1 s. The 1 s time constraint is chosen to mimic the time-limited nature of cardiac contractions, where the exposure to low calcium concentration solutions mimics the diastolic phase and the exposure to high calcium concentration solutions mimics the systolic phase of cardiac muscle contraction (Figure 11A). For each of the five sets diastolic calcium was varied (1, 80, 160, 250, and 400 nM calcium), while systolic calcium remained constant (Figure 11A). A set consisted of two sets of three activation-relaxation cycles at 1.8 &#181;m versus 2.0 &#181;m and 2.0 &#181;m versus 2.2 &#181;m for different experimental groups. Peak force was measured at 1 s from the switch of the pipette and averaged for the set of three activation-relaxation cycles. The high signal-to-noise ratio and the high dynamic range of this force transducer allowed us to measure both the small changes in diastolic force and the much larger systolic forces (Figure 11B). Increasing diastolic calcium resulted in a higher force at 2 &#181;M calcium relative to the first activation (Figure 11B). WT rat cardiomyocytes were compared with heterozygous (HET) RMB20 rat cardiomyocytes. Due to alternative splicing, HET rats have a more compliant titin protein as compared to the WT rats. The effect was exaggerated in HET cardiomyocytes at 80 and 160 &#181;M calcium (Figure 11C).

Striated muscle cells are indispensable for daily life activities. Limb movement, respiratory function, and the pumping motion of the heart rely on the force generated by muscle cells. Skeletal muscle consists of muscle fascicles containing bundles of single muscle fibers (Figure 1A). These muscle fibers are comprised of myofibrils, which are formed by serially linked sarcomeres (Figure 1B,D). The sarcomeres contain thin and thick filaments. These primarily consist of chains of actin and myosin molecules, respectively (Figure 1B). Actin-myosin interactions are responsible for the force-generating capacity of muscle. Patients with mutations in genes encoding for sarcomeric proteins, such as nebulin, actin, and troponin T, suffer from muscle weakness due to contractile dysfunction1.
The quality of muscle contractility can be studied at various levels of organization, ranging from in vivo whole muscles to actin-myosin interactions in in vitro motility assays. During the past decades, several research groups have developed setups to determine the contractility of individual myofibrils2,3,4,5,6,7,8,9,10. These setups are based on the detection of changes in laser deflection from a cantilever (i.e., optical beam deflection) caused by the contraction of the myofibril (for details, see Labuda et al.11). Although determining the contractile function of myofibrils has some limitations (e.g., the dynamics of the excitation-contraction coupling processes that are upstream of the myofibrils are lacking), there are multiple advantages to this approach. These include: 1) the ability to assess actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres; 2) the ability to assess actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils (when measuring the contractility of single muscle fibers ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility) (Figure 1D); 3) the small diameter of myofibrils (~1 &#181;m, Figure 2A) and the lack of membranes allow for almost instant calcium diffusion into the sarcomeres. Furthermore, if structural damage is present in the myofibrils, they likely break during their isolation or during the experiment. Hence, assessing myofibril contractility is an elegant method to study the basic mechanisms of muscle contraction and to understand whether disturbed actin-myosin interactions are the primary cause of muscle disease caused by mutations in sarcomeric proteins.
This protocol presents a newly developed setup to determine the contractility of myofibrils incorporating a cantilever force probe with nano-Newton resolution (i.e., Optiforce). This force probe is based on the principle of interferometry. Interferometry enables the use of relatively stiff cantilevers. This makes it possible to measure force with little deflection of the cantilever, approaching isometric contractions of the myofibril. The probe allows for the assessment of low passive and active forces that are produced by a single myofibril isolated from different muscle biopsies, including those from human subjects, with a high signal-to-noise ratio. The optical cantilever force probe incorporated in this setup is based on a Fabry-P&#233;rot interferometer12. The interferometer detects small displacements between an optical fiber and a gold-coated cantilever mounted on a ferrule (Figure 3). The gap between the optical fiber and the cantilever is called the Fabry-P&#233;rot cavity. Myofibrils are mounted between the probe and piezo motor using two glue-coated glass mounting fibers. The force produced by the myofibril can be mathematically derived from the interferometer data. Interferometry is based on the superposition or interference of two or more waves (in this setup three light waves). Laser light with a wavelength between 1,528.77&#8211;1,563.85 nm is emitted from the interferometer and is sent through the optical fiber. In the probe, the light is reflected 1) at the interface between the optical fiber and the medium (Figure 3A); 2) at the interface of the medium and the cantilever (Figure 3B); and 3) at the interface between the metal and gold coating of the cantilever (Figure 3C). The reflection at interface A and B is dependent on the refractive index (n) of the medium in which the probe is submerged. The light, consisting of the three superimposed reflections, returns to a photodiode in the interferometer. The photodiode measures the intensity of the light, which is the result of the interference pattern of the three superimposed reflections. When contractile force is generated by activating or stretching a myofibril, the myofibril pulls on the cantilever. This movement changes the cavity size (d) and consequently, the number of wavelengths that fit in the cavity. The light reflected at the cantilever will have a different phase, resulting in a different interference pattern. The photodiode records this change of interference pattern intensity as a change in Volts. Subsequently, myofibril force generation is calculated from this change, taking into account the cantilever stiffness. The force probe is calibrated by the manufacturer by pushing the tip of the mounting needle, attached to the free handing end of the cantilever, against a weighing scale while keeping the bending of the cantilever equal to a multiple of the wavelength of the readout laser13. Thus, interferometry is a highly sensitive method to detect small changes in distance, allowing for measurement of forces with nano-Newton resolution. This resolution enables the assessment of myofibrillar force production with a high signal-to-noise ratio. While traditional interferometry limits the range of measurements to the linear part of the interference curve, using a lock-in amplifier and modulation of the laser wavelength overcomes this limitation14. This is explained in more detail in the discussion section.
To measure myofibril active tension, a fast-step perfusion system was incorporated to expose the myofibril to calcium solutions (Figure 4A). The fast-step perfusion system enables solution changes to occur within 10 ms. Because of their small diameter, calcium diffusion into the myofibrils is nearly instantaneous. Hence, this system is particularly suitable for measuring the rates of actin-myosin binding during activation and release during relaxation. The rate of activation (kACT) and relaxation (kREL) can be determined from the activation-relaxation curves. Also, by exposing the myofibrils to calcium solutions of increasing concentration, the force-calcium relationship and calcium sensitivity can be determined.
Furthermore, a piezo length motor enables fast stretching and shortening of the myofibril. This offers the possibility to study the viscoelastic properties (i.e., passive tension) of the myofibril, as well as performing a rapid shortening and restretch of the myofibril to determine the rate of tension redevelopment (kTR). The parameters retrieved from both active and passive tension experiments can be altered by gene mutations in a sarcomeric protein.
This custom-built setup was used to measure the active and passive contractile characteristics of myofibrils isolated from healthy human, patient, and mouse skeletal muscle.

<table><tbody><tr><td>Bio Spec Products, Inc.</td><td>985370-XL</td><td>To isolate myofibrils</td><td></td></tr><tr><td>Custom coded</td><td></td><td>Matlab</td><td></td></tr><tr><td>Custom fabricated</td><td></td><td>Includes Labview program to control over serial connection; To control valves</td><td></td></tr><tr><td>Custom fabricated</td><td></td><td>To cool the Peltier module</td><td></td></tr><tr><td>Custom fabricated</td><td></td><td></td><td></td></tr><tr><td>Custom fabricated</td><td></td><td>Aluminum tissue chamber</td><td></td></tr><tr><td>Custom fabricated</td><td></td><td>To control the valves; Includes PC software to control over USB</td><td></td></tr><tr><td>IonOptix</td><td></td><td>System controller software: data recording software with advanced signal generator for piezo and fast-step</td><td></td></tr><tr><td>IonOptix</td><td>MCS100</td><td>To record sarcomere length</td><td></td></tr><tr><td>IonOptix</td><td></td><td>Includes: Optiforce (interferometer), Micromanipulators, Signal interface, Piezo motor and controller. Based on the MyoStretcher</td><td></td></tr><tr><td>IonOptix</td><td></td><td>Force probe</td><td></td></tr><tr><td>Koolance</td><td>ADT-EX004S</td><td></td><td></td></tr><tr><td>Koolance</td><td>EX2-755</td><td>To cool the Peltier module</td><td></td></tr><tr><td>Microsoft</td><td></td><td>Data registration</td><td></td></tr><tr><td>Olympus</td><td>IX71</td><td></td><td></td></tr><tr><td>Olympus</td><td>TH4-200</td><td></td><td></td></tr><tr><td>Sigma-Aldrich</td><td>529265</td><td>Poly(2-hydroxyethyl methacrylate); Coating for microscope slides to prevent sticking of tissue</td><td></td></tr><tr><td>Sigma-Aldrich</td><td>78471</td><td>Crystals to dissolve in ethanol resulting in glue</td><td></td></tr><tr><td>TE Technology, Inc.</td><td>TE-63-1.0-1.3</td><td>To cool the tissue flow chamber</td><td></td></tr><tr><td>TE Technology, Inc.</td><td>TC-720</td><td>Includes PC software to control over USB</td><td></td></tr><tr><td>Tecan Trading AG</td><td>20736652</td><td></td><td></td></tr><tr><td>Tecan Trading AG</td><td>20739263</td><td>Syringe pump to induce backgroundflow together with fast-step perfusion system; Outflow from tissue flow chamber</td><td></td></tr><tr><td>Thermo scientific</td><td>2441081</td><td></td><td></td></tr><tr><td>Warner Instruments (Harvard Bioscience, Inc.)</td><td>Discontinued</td><td>Alternative: SF-77CST/VCS-77CSP</td><td></td></tr><tr><td>Warner Instruments (Harvard Bioscience, Inc.)</td><td>TG150-4</td><td>To perfuse the tissue</td><td></td></tr><tr><td></td><td></td><td>1 PC for IonWizard and 1 PC for other software</td><td></td></tr></tbody></table>

isolating myofibrils from skeletal muscle biopsies and determining contractile function with a nano-newton resolution force transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Watch this Scientific Journal Video about Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer at JoVE.com

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

isolating-myofibrils-from-skeletal-muscle-biopsies-determining

Nanyang Technological University

Research

JoVE Journal

Biology

6.9K Views.  Amsterdam UMC. Het beoordelen van de contractiele eigenschappen van myofibrils geïsoleerd van gestreepte spieren kan worden gebruikt om te bepalen of sarcomere disfunctie is een primaire oorzaak van spierzwakte als gevolg van mutaties in sarcomerische eiwitten. Deze techniek kan worden gebruikt om gegevens te verkrijgen met een zeer hoge signaal-ruisverhouding met nanonewtonresolutie. Dit protocol is ook geschikt om contractiliteit te meten in permeabiliseerde cardiomyocyten.Houd er rekening mee dat...

Video: Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Bioengineering

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

In Vitro Assessment of Cardiac Function Using Skinned Cardiomyocytes

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring apparatus and a motor to perform functional studies. These studies will allow measurement of cardiomyocyte stiffness (passive force) and its activation with different calcium (Ca2+)-containing solutions to determine, amongst others: maximum force development, myofilament Ca2+-sensitivity (pCa50), cooperativity (nHill) and the rate of force redevelopment (ktr). This method also enables determination of the effects of drugs acting directly on myofilaments and of the expression of exogenous recombinant proteins on both active and passive properties of cardiomyocytes. Clinically, skinned cardiomyocyte studies highlight the pathophysiology of many myocardial diseases and allow in vitro assessment of the impact of therapeutic interventions targeting the myofilaments. Altogether, this technique enables the clarification of cardiac pathophysiology by investigating correlations between in vitro and in vivo parameters in animal models and human tissue obtained during open heart or transplant surgery.

This protocol aims to describe step-by-step the technique of extraction and assessment of cardiac function using skinned cardiomyocytes. This methodology allows measurement and acutemodulation of myofilament function using small frozen biopsies that can be collected from different cardiac locations, from mice to men.

In this article, we describe the steps required to isolate a single permeabilized (&#34;skinned&#34;) cardiomyocyte and attach it to a force-measuring ...

Medicine

DNA Electroporation, Isolation and Imaging of Myofibers

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently labeled proteins in the flexor digitorum brevis (FDB) muscle. Conditions have been optimized to provide a large number of high quality myofibers expressing the electroporated plasmid while minimizing muscle damage. The method employs fluorescent tags on various proteins. Combining this expression method with high resolution confocal microscopy permits live cell imaging, including imaging after laser-induced damage. Fluorescent dyes combined with imaging of fluorescently-tagged proteins provides information regarding the basic structure of muscle and its response to stimuli.

This protocol utilizes electroporation to introduce and express fluorescently labeled proteins in mouse muscle fibers. Following recovery after electroporation, fibers are isolated. Individual fibers are then imaged using high resolution confocal microscopy to visualize muscle structure.

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

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