Name: isolating myofibrils from skeletal muscle biopsies and determining contractile function with a nano-newton resolution force transducer
Uploaded: 2020-05-07T14:00:00
Description: 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

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 ...

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