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

Summary

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.

Abstract

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-Pérot 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.

Introduction

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 re....

Protocol

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 concentration (pCa) solutions^7,16,17, and isolate myofibrils

Results

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.......

Discussion

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-Pérot 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 (.......

Materials

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