Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

The overall goal of this procedure is to measure the maximum isometric force generating capacity of individual fibers from chemically perme skeletal muscles samples. This is accomplished by first exposing the muscle fiber membranes to a chemical detergent, thereby removing the barrier between extracellular and intracellular contents. The second step is to extract an individual fiber from a small bundle of fibers and attach it to the experimental apparatus.

Next, laser interference patterns are used to set the optimal sarcomere length for the individual fiber. Finally, calcium is administered to elicit maximum isometric contraction force. Ultimately, the perme single fiber technique provides an assessment of the contractile properties of skeletal muscle without the complexities associated with whole muscle architecture.

Visual demonstration of this method is useful as the key steps to performing the protocol successfully can be difficult to learn. Start by preparing the dissection, storage, and testing solutions as described in the accompanying text protocol. Next, use forceps to prepare suture loops from strands of USP 10 zero monofilament nylon suture.

Using the double overhand knot technique, prepare four loops for every fiber to be tested. Once formed, place the knot under a microscope and reduce its size to approximately 750 micrometer diameter. Using the IPS GTI markings as a guide.

Then trim any excess suture, leaving only the loop and small 500 micrometer tails on either side. Transfer a small mass of muscle tissue obtained via open or needle biopsy to a dish containing chilled dissection solution and add more dissection solution to ensure that the tissue remains submerged. Inspect the sample under the microscope and manipulated to align the longitudinal axis of the fibers to towards the right shoulder of the investigator.

Then anchor the sample to the dish by pinning it at the corners with the forceps in the left hand and the micro dissecting scissors in the right. Begin gently dissecting a bundle along the longitudinal margins between fibers. Remove and discard any tissue that is damaged by the forceps or pins.

As a result of the dissecting process, transfer the bundles from the dissecting solution into a three milliliter vial containing 2.5 milliliters of fresh chilled dissecting solution with a non ionic detergent added. Incubate the sample on ice for 30 minutes with occasional gentle agitation and ensure that the bundles remain submerged throughout incubation. Then transfer the bundles to a vial containing fresh dissecting solution and agitate gently and briefly to remove any remaining detergent.

Once rinsed, transfer the bundles to a vial containing chilled storage solution and incubate overnight at four degrees Celsius. The next day, remove the vial from storage at four degrees Celsius and transfer all bundles to a medicine cup or dissecting dish. In preparation for storage, transfer the bundles into individually labeled cryo vials, filled with 200 to 400 microliters of fresh storage solution, and ensure that the bundle is not stuck to the side of the tube or floating on the surface.

Then cap the tube and store the sample at negative 80 degrees Celsius. Set up an experimental apparatus like the one shown here and ascribed in the accompanying text protocol. Then fill the first experimental chamber with relaxing solution, the second chamber with pre reactivating solution, and the third with activating solution.

Adjust the temperature in the chamber until it is steady at 15 degrees Celsius with the apparatus in the relaxing solution. Thread two of the prepared suture loops onto the stainless steel attachment surfaces extending from both the force transducer and the length controller. Next, thaw a fiber bundle of interest on ice for about 15 minutes, and then transfer it to a silicone elastomer plated Petri dish filled with fresh, chilled relaxing solution.

Secure the bundle in place with pins at either end and ensure that it is submerged using the forceps. Grasp a fiber at one end and begin smoothly extracting it along its longitudinal axis. Care should be taken when extracting fibers from the bundle since adhesions between the fibers and the extracellular matrix may result in excessive tension, ultimately leading to stretch induced damage.

Once extracted, introduce the fiber into a modified pipette tip along with a small amount of relaxing solution and transfer it to the experimental chamber that contains the relaxing solution guiding gently with the forceps. Remove the fiber from the pipette tip and anchor it to the length controller. Using the first suture loop, manipulate the other end of the fiber toward the force transducer and secure the fiber using the same procedure.

Remove any excess suture using the micro dissecting scissors. Next, thread the second loop over the first and anchor the fiber at a point within 0.2 millimeters of the end of the length controller attachment surface. Repeat with the second loop at the force transducer end of the fiber.

Adjust the position of the length controller in the direction of the Y axis in order to align the fiber parallel to the side walls of the experimental chamber. Then survey the fiber using the prism side view and adjust position of the length controller in the direction of the Z axis until the fiber is parallel to the floor of the chamber. If the fiber is damaged in any way, the fiber should be discarded and a new fiber attached.

Turn on the laser and adjust the position of the stage such that the laser passes through the center of the fiber. Then set the sarcomere length by increasing or decreasing the tension on the fiber using the length controller X-axis micrometer driver until the desired spacing of the diffracted light is observed on the target screen. After optimal sarcomere length has been set, measure the distance between the two innermost sutures.

Position the chamber such that the vertical cross hair of the eyepiece is aligned on the innermost border of one inside suture and zero. The digital readout on the micrometer drive translate the stage along the x axis relative to the microscope until the vertical cross hair of the eyepiece is aligned with the innermost suture. The digital display will indicate the length of the fiber, maintain the fiber at the measured length, and use the microscope mounted camera to capture a high magnification image of the central portion of the fiber from both the top and side views.

Use these images to calculate the cross-sectional area of the fiber. Use the chamber control software to move the fiber to the chamber containing the pre reactivating solution and incubate there for three minutes. While the fiber is inactivating solution, measure its passive tension, then move the fiber to the chamber containing the activating solution and allow maximum isometric force to develop as evidenced by a plateau in force that is preceded by a rapid rise.

Next, use the length controller to apply a large rapid and brief reduction in the distance between the fiber attachment points, followed by a rapid return to the original length to identify the force transducer output that corresponds to zero. Force shown here are both the top and side views of an elliptically shaped fiber from these views. The cross-sectional area was determined by measuring the diameters at five points along the length of the fiber and then calculating the average of the five resulting areas.

Force traces from human, slow and fast muscle fibers illustrate that fast fibers generate force at a higher rate than slow fibers using the technique shown in this video. The typical characteristics of human mouse and rat muscle fibers were measured and are presented here. The range shown indicates the 25th to 75th quartiles and the N is the number of fibers tested.

Note the wide variation in specific force among the different species. The procedures outlined here result in the assessment of the maximum intrinsic force generating capability of a single skeletal muscle fiber by adding additional interventions, important aspects of skeletal muscle physiology, including among others, length force, force velocity, and force. Calcium relationships can be revealed.