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.

The protocol for obtaining human biopsies was approved by the institutional review board at VU University Medical Center (#2014/396) and written informed consent was obtained from the subjects. The protocol for obtaining animal muscle biopsies was approved by the local animal ethics committee at VU University (AVD114002016501)
1. Preparation and myofibril isolation
NOTE: Use previously described methods to glycerinate biopsies, prepare the different calcium concentrat...

<ol>
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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 (...

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.

Data traces were recorded and opened with the system controller software (see Table of Materials). Complete traces or selected segments were exported to the clipboard or text file for further analysis with a desired software. Valves to control flow of the different solutions were switched with custom software or manually. A custom MATLAB script was used to analyze the rates of activation, tension redevelopment, and relaxation. The maximum active force and the peak and the plateau force of the passive for...

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 ...

Assessing the contractile properties of myofibrils isolated from striated muscle can be used to determine whether sarcomere dysfunction is a primary cause of muscle weakness due to mutations in sarcomeric proteins. This technique can be used to acquire data with a very high signal-to-noise ratio with nanonewton resolution. This protocol is also suitable to measure contractility in permeabilized cardiomyocytes.Keep in mind that the force transducer is very fragile. So when removing the glue from the mounting needle or when gluing a myofibril, you need to have a steady hand, a lot of practice, and a lot of patience. Before mounting the tissue, place a homogenizer rod in the tube containing the muscle tissue.Keeping the tube on ice, spin the rotor for 15 seconds on speed five. At the end of the homogenization, transfer 50 microliters of the resulting myofibril suspension and 250 microliters of relaxing solution onto a poly-HEMA coated slide in a tissue bath. Cover the bath with a lid to protect the mixture from dust and wait 5 to 10 minutes to allow the myofibrils to sink to the bottom of the drop.During the sinking of the myofibrils, heat Shellac an ethanol glue at 65 degrees Celsius for 30 to 60 seconds before adding about six microliters of glue onto an uncoated glass slide. Repeatedly dip the tip of each mounting needle into the glue until a layer of glue is visible. Then use micro-manipulators to move the probe and piezo vertically to make room for the tissue bath on the microscope stage and remove the glass slide containing the glue.After mounting the myofibrils, to measure the sarcomere length, move the piezo and/or force probe to set the initial sarcomere length of the myofibril to 2.5 micrometers. Using the vessel function of the system controller software, measure the myofibril length and width. Use the microscope stage to position the myofibril in the center of the video image and stretch a rectangle from one side of the myofibril to the other, taking care to include the dark edge of the glue droplets.To start recording the data, click start. After five seconds, click pause. The length will have been recorded.To measure the width, rotate the camera 90 degrees to view the contrast of the edge of the myofibril itself. Adjust the rectangle and click start to start recording the data. After five seconds, click pause.The width will have been recorded. To position the theta glass, use the eyepiece and the manipulator to carefully move the theta glass toward the myofibril. Align the top channel of the theta glass with the myofibril and perform a fast-step to check the position.Turn on the background relaxing flow to check the theta glass alignment and use a Luer valve lever to turn on the inflow of the flow chamber. Then to start draining the flow chamber and to prevent overflowing of the flow chamber, set the outflow pump valve to bath valve two, the micro step mode to micro, the plunger target to 48, 000, and the plunger speed to 38 to 40. To measure the rate of tension redevelopment, calculate the piezo movement necessary to slacken the myofibril by 15%and enter this value into the signal generator.Click resume to continue recording the data and open valves one and six to start the flows of the relaxing solution and the different calcium concentrations through the theta glass respectively. Select reset range on the interferometer to reset the range of the interferometer so that the baseline force is zero volts. When the force trace is stable, perform the theta glass fast-step with a 100 micrometer step size.When the force plateau is reached, perform the shortening re-stretch with the piezo. An activation relaxation trace will be recorded. Click pause.To perform a step-wise stretch, click start and reset the range of the interferometer so that the baseline force is zero volts. Perform a step-wise stretch with the signal generator. When the stretch is finished, use the piezo to shorten the myofibril to the slack length.Then press pause and stop and save the data. Here, force traces of an active force experiment with a myofibril isolated from healthy human quadriceps muscle are shown. The myofibril was activated five times with solutions with varying calcium concentrations, with an average maximum force of all of the myofibrils of approximately 123 millinewtons per square millimeter.The construction of a force calcium concentration curve from the plateau forces reached during each activation in each of the five calcium curves allows calculation of the calcium concentration at 50%of the maximum force production. As illustrated in this representative analysis, the myofibrils can be treated with multiple compounds in a single experiment to answer additional questions about the myofibril type. The active force and sarcomere length can also be measured to allow calculation of the rates of redevelopment, activation and relaxation.The steep rise shown in the dotted red box is caused by both viscosity and elasticity. The plateau resembles only the elastic component. As viscosity resists strain linearly, the force dropped after the strain was removed.When positioning the theta glass, make sure you align it properly with the myofibril. Otherwise, the activating solution might come in between the optical fiber and the cantilever of the force probe and this causes artifacts to occur in your data. Also, the myofibril may not activate properly.This method of perfusion is also suitable to determine the muscle fiber type of the myofibril. For example, you can first perfuse the myofibril with a normal calcium solution followed by perfusion of that same solution, but then add a muscle fiber-type specific force-enhancing compound.

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

Research

JoVE Journal

Biology

6.8K 조회수.  Amsterdam UMC. 수축된 근육으로부터 분리된 myofibrils의 수축 특성을 평가하는 것은 sarcomere 기능 장애가 육종 단백질의 돌연변이로 인한 근육 약화의 주요 원인인지 여부를 결정하는 데 사용될 수 있습니다. 이 기술은 나노 뉴턴 해상도를 가진 매우 높은 신호 대 잡음 비율로 데이터를 획득하는 데 사용할 수 있습니다. 이 프로토콜은 또한 투과성 심근세포에서 수축을 측정하기 위하여 적당?...