Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

All procedures involving animal or human subjects should be performed in accordance with relevant guidelines, regulations, and regulatory agencies. The University of Michigan Committee on the Use and Care of Animals (UCUCA) and the University of Michigan Medical Center Institutional Review Board approved all animal and human procedures described in this article.

1. Make Dissecting and Storage Stock Solution

Note: The final volumes specified in the following instructions can be scaled up or down as desired.

1. In a 1,000 ml beaker add 800 ml of deionized water (ASTM Type 1). Maintaining a gentle stir, add all the compounds listed in Table 1 to the deionized water and allow them to dissolve.

   Compound	Desired Conc. (M)	Formula Weight (g/mol)	Add to 1 L (g)
   K-propionate	0.250	112.17	28.040
   Imidazole	0.040	68.08	2.720
   EGTA	0.010	380.40	3.800
   MgCl2•6H2O	0.004	203.31	0.813

Table 1: Dissecting and storage stock solution components.

2. Bring to a final volume of 1,000 ml with deionized water. Note that it is unnecessary to pH the solution at this time. Store the stock solution at 4 °C.

2. Make Storage Solution

1. To make 200 ml of storage solution, begin with 100 ml of dissecting and storage stock solution in a 250 ml beaker. Add sufficient Na₂H₂ATP to bring final adenosine triphosphate (ATP) concentration to 2 mM. Bring to a final volume of 200 ml with glycerol. Adjust to pH 7.00 with potassium hydroxide (KOH). Store the storage solution at -20 °C.
   Note: Due to the viscous nature of glycerol it can be difficult to accurately dispense by volume. Because of this, we typically add the glycerol by weight (100 ml of glycerol weighs approximately 126 g).

3. Make Dissecting Solution

1. To make 200 ml of dissecting solution, begin with 100 ml of the dissecting and storage stock solution in a 250 ml beaker.
2. Add sufficient Na₂H₂ATP to bring final ATP concentration to 2 mM. Adjust pH to 7.00 with KOH. Bring the final volume to 200 ml with deionized water. Aliquot in volumes of 2.5 ml and store at -80 °C.

4. Make Dissecting Solution with Brij 58

Note: Brij 58 is a non-ionic detergent that disrupts (permeabilizes) lipid bilayers.

1. To make 200 ml of dissecting solution with Brij 58, begin with 200 ml of the dissecting solution in a 250 ml beaker. Maintaining a gentle stir, add 1 g of Brij 58 (0.5% w/v) to the dissecting solution and allow it to dissolve. Aliquot in volumes of 2.5 ml and store at -80 °C.

5. Make Testing Solutions

Note: The following is adapted from Moisescu and Thieleczek 1978 (6). See Discussion for additional comments on preparing testing solutions.

1. Prepare three separate 1,000 ml beakers labeled “Relaxing”, “Pre-activating” and “Activating”. Add 400 ml of deionized water to each beaker.
2. Add the compounds indicated in Table 2 to the appropriate beaker and then heat the solutions to between 70 °C and 80 °C. Maintain a solution temperature of 70-80 °C for 30 min while stirring continuously.
   Note: A temperature of 70-80 °C assists in the elimination of carbonic acid formed by the reaction of calcium carbonate with EGTA in the activating solution. The relaxing solution and pre-activating solutions are treated in the same way as the activating solution to maintain consistency.

Table 2: Relaxing, pre-activating and activating solution components.

3. Cool the solution to room temperature and add sufficient NaN₃/KOH to bring the final NaN₃ concentration to 1 mM.
   CAUTION: NaN₃ (sodium azide) is poisonous. Refer to the chemical MSDS prior to handling this chemical.
   1. To make 100 ml of 100 mM sodium azide solution, add 0.65 g of NaN₃ to 10 ml of 1 N KOH. Adjust to a final volume of 100 ml with deionized water.
4. Adjust the pH to approximately 7.10 using KOH.
5. Following step 5.4 add sufficient Na₂H₂ATP to bring the final ATP concentration to 8 mM and sufficient Na₂CrP to bring the final creatine phosophate (CrP) concentration to 10 mM.
6. Bring each solution to the final volume of 500 ml using deionized water. Chill or heat the solutions to the temperature at which experiments will be performed, then use KOH to bring the pH to 7.10 while maintaining that temperature.
7. Add relaxing solution to the beaker containing the pre-activating solution such that the final pre-activating solution is 1 part relaxing solution in 500 pre-activating solution. Aliquot in volumes of 2.5 ml and store at -80 °C.

6. Make Suture Loops

1. Begin with a strand of non-sterile USP 10-0 monofilament nylon suture.
2. Use the forceps to create a loop with the strand using the double overhand knot technique. Reduce the knot in size to approximately 750 µm diameter. Loop diameter can be assessed under the microscope using eyepiece graticule markings.
3. Use microdissecting scissors to remove excess suture leaving only the loop and small (500 µm) tails on either side. An example of a finished loop is shown in Figure 1.
4. Repeat steps 6.2-6.3 until 4 usable loops have been made. Store loops in a silicone elastomer-plated petri dish for future use.
   Note: Four suture loops are required for every fiber tested.

7. Bundle Sample

Note: The following steps describe the procedure for dissecting the original sample into smaller experimental ‘bundles’ from which single fibers will eventually be extracted and tested. At all times the sample should be treated with care. For the purpose of this description, instructions will be given as if the investigator is right handed.

1. Obtain the sample of interest and transfer it to the facility where dissection will take place.
   Note: Methods of tissue biopsy will vary depending on experimental model and study design. Where possible, muscle perfusion should be maintained up until the time of the biopsy.
2. If the sample is to be transferred between the harvest site and the dissecting site, transport it in a vial containing chilled dissecting solution while maintained on ice.
3. Prepare a 5 cm silicone elastomer-plated petri dish with chilled dissecting solution and two to three insect mounting pins (100 µm diameter, stainless steel).
4. Transfer the sample to the dish. Ensure that the sample remains submerged by adding more dissecting solution if necessary.
5. Inspect the sample under the microscope and manipulate it to align the longitudinal axes of the fibers toward the right shoulder of the investigator (Figure 2). Then anchor the sample to the dish by pinning at the corners.
   Note: Make use of any remaining connective tissue as anchoring points at this time since this will maximize sample use and conserve fiber integrity. The bundle may be pinned in slight tension to assist in defining inter-fiber margins.
6. With the forceps in the left hand and the microdissecting scissors in the right, begin gently dissecting a bundle along the longitudinal margins between fibers
   Note: Depending on its overall length, further dissect the bundle into numerous smaller bundles.
7. Ensure that bundle dimensions measure approximately 0.5-1 mm in width and ≥3 mm in length. Assess the dimensions using a microscope with graticule markings in the eyepiece, or by placing a ruler under the dish.
8. Remove and discard any tissue that is damaged by the forceps or pins as a result of the dissecting process.
9. Repeat the process until a sufficient number of bundles have been dissected or until the sample has been exhausted.
   Note: The number of bundles that can be obtained will depend on numerous factors including the size and condition of the initial sample, the morphology of the muscle, and the skill of the investigator.

8. Permeabilize Fibers

1. Transfer the bundles from the dissecting solution into a vial containing 2.5 ml of fresh, chilled, dissecting solution with the non-ionic detergent ‘Brij 58’ added (0.5%, w/v). Incubate on ice for 30 min with occasional, gentle agitation. Ensure that the bundles remain submerged throughout incubation.
2. At the end of the 30 min incubation, transfer the bundles to a vial containing fresh dissecting solution (no Brij 58) and agitate gently and briefly to remove any remaining detergent.

9. Prepare Bundles for Storage

1. Transfer the bundles to a vial containing chilled storage solution and incubate overnight at 4 °C.

10. Store Bundles

1. The following day, prepare a storage box, capable of withstanding -80 °C, with enough individual 0.5 ml screw cap conical tubes to accommodate all bundles obtained during the dissecting process (one bundle per tube). Each conical tube should be filled with 200-400 µl of fresh storage solution.
2. Transfer the bundles into the individually labeled conical tubes. Ensure that the bundle is not stuck to the side of the conical tube or floating on the surface of the solution. Cap the conical tubes and store the samples at -80 °C until the day of testing.

11. Prepare Experimental Apparatus

Note: The custom apparatus is composed of a stage that houses a length controller and force transducer, a moving chamber system and a 10X dissection microscope. Micrometer drive installations allow for precise manipulation of fiber attachment surfaces. Laser diffraction patterns are used to estimate sarcomere length. Data generated during experimentation is recorded on a personal computer. Refer to Figure 3 for annotated images of the experimental set-up.

1. Thaw one vial each of the relaxing, pre-activating and activating solutions and maintain on ice. Note that ATP and CrP are labile compounds that should be maintained at cold temperatures.
2. Prepare the microscope, testing apparatus and associated computer for use.
3. Fill the first experimental chamber with relaxing solution. In our apparatus, the first chamber contains prisms that allow the investigator to photograph the fiber from the side. Fill the second experimental chamber with pre-activating solution and the third with activating solution.
4. Adjust the temperature so that the in-chamber thermometer display reads 15 °C. Thread two prepared suture loops onto the stainless steel attachment surfaces extending from both the force-transducer and length-controller (Figure 5A).

12. Extract Permeabilized Single Fiber

1. Thaw a fiber bundle of interest, and transfer to a silicone elastomer-plated petri dish with fresh, chilled relaxing solution. Secure the bundle with pins at either end and ensure that it is submerged.
2. Using the forceps, grasp a fiber at one end and begin smoothly extracting it along its longitudinal axis.
   Note: Compressive damage to the end of the fiber caused by the forceps is not a concern at this time since the contractile properties in this area will not be tested. Care should, however, be taken when extracting fibers from the bundle since adhesions between fibers and the extracellular matrix may result in excessive tension, ultimately leading to stretch-induced damage. Note that considerable variability exists among muscles in the degree to which the fibers adhere to the surrounding extracellular matrix. Fibers that are suspected to have been damaged by such a stretch should be discarded.
3. Use a razor blade or scalpel to modify a pipet tip as shown in (Figure 4). Introduce the fiber into the tip along with a small amount of relaxing solution. Transfer the single fiber from the silicone elastomer-plated petri dish to the experimental chamber that contains the relaxing solution.

13. Mount Single Fiber

Note: A step-by-step depiction can be viewed in Figure 5.

1. Guiding gently with the forceps, remove the fiber from the pipet tip and anchor it to the length-controller (left) using the first suture loop.
   1. Use a single, smooth motion when tightening the loop with the forceps. Ensure that an equal and opposite tension is applied to either end of the suture.
   2. The first loop should be tied around 1 mm to 2 mm from the end of the length-controller attachment surface.
2. Manipulate the other end of the fiber toward the force-transducer (right) and secure the fiber using the same procedure. Remove excess suture using the microdissecting scissors (Figure 5C).
3. Place the fiber under a small amount of tension by increasing the distance between the length-controller and force-transducer arms using the x-coordinate micrometer drive (Figure 3A).
4. Thread the second loop over the first and anchor the fiber at a point within 0.2 mm of the end of the force-transducer attachment surface (Figure 5D).
   1. Remove excess suture using the microdissecting scissors.
5. The fiber attachment process can result in loss of solution from the chamber. If necessary, add more relaxing solution to ensure the surface of the solution is flat (neither concave nor convex). A flat surface is important when assessing sarcomere length using laser diffraction.
6. Align the fiber parallel to the sidewalls of the experimental chamber by adjusting the position of the length-controller in the direction of the y-axis.
7. Survey the fiber using the prism side view and adjust the position of the length-controller in the direction of the z-axis until the fiber is parallel to the floor of the chamber.
   Note: Positioning of the fiber parallel to the floor of the chamber can be accomplished without chamber prisms by focusing first on one end of the fiber and then, without adjusting the microscope focus, bringing the other end of the fiber into focus using its z-axis micrometer drive.
8. If the fiber is in any way contorted, twisted or damaged as a result of the mounting process, the fiber should be discarded and a new fiber attached.

14. Set Optimal Sarcomere Length

1. When the fiber has been correctly aligned within the chamber, insert the calibrated target screen onto the anterior aspect of the microscope and align it over the first experimental chamber.
   Note: The target screen is calibrated using the standard grating equation, λ = SL sinθ, where SL is sarcomere length, θ is the diffraction angle between the 0° and 1° diffracted beams and λ is the wavelength of the laser.
2. Turn on the laser and adjust the position of the stage such that the laser passes through the center of the fiber.
   CAUTION: Concentrated laser light can be damaging to eyesight. Never attempt to visualize the fiber through the microscope when the laser is on.
3. Position the fiber with respect to the laser beam to diffract the laser light and observe an interference patter on the calibrated target screen (Figure 6). Turn off the lights to visualize this pattern more clearly.
   Note: If, with the correct positioning of the laser beam, no interference pattern is seen, this suggests that the myofibrillar components of the fiber are abnormal/damaged and that the fiber should be replaced with a fresh one.
4. To set the sarcomere length, increase or decrease the tension on the fiber using the length-controller x-axis micrometer drive until the desired spacing of the diffracted light is observed on the target screen.
   Note: The optimal sarcomere length of the fiber will depend on the species of animal from which the sample was obtained. A sarcomere length of 2.7 µm is commonly assumed to be optimal when assessing fibers from human tissue^7,8.
5. After optimal sarcomere length has been set, measure the distance between the two innermost sutures. This is most easily accomplished using the digital readout on the micrometer drive that controls the x-axis motion of the chamber. Position the chamber such that the vertical cross hair of the eyepiece is aligned on the innermost border of the innermost suture and zero the digital readout on the micrometer drive.
6. Translate the stage along the x-axis relative to the microscope until reaching the other innermost suture. The digital display will indicate the length of the fiber. This value should be recorded as fiber length, L_f.
   Note: It should be understood that the distance between the two innermost sutures will determine the functional length of the contractile tissue being evaluated. The investigator should strive for consistency in this dimension (i.e. L_f) within a series of experiments.

15. Estimate Cross-sectional Area (CSA)

1. Maintain the fiber at L_f 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. Side view images can be captured using the prism embedded in the side of the chamber.
   Note: When shifting between top and side views it is important to move the microscope only in the y-direction to ensure that the two images are “in register” and therefore show two different views of the same section of the fiber.
2. Obtain measurements at this time to normalize absolute force later in the study. The techniques for obtaining these measurements are described later and illustrated in Figures 7A and 7B.

16. Elicit Isometric Contraction

Note: While data generated during these experiments may be collected and interpreted without the use of a computer, software that allows for the acquisition, display, storage and analysis of the force responses is advantageous. The custom LabVIEW software created by our laboratory allows these functions as well as the capability to design ‘motion trains’ that govern the action of the length-controller during an experiment.

1. Confirm that the temperature of the relaxing, pre-activating and activating solutions are stable at 15 °C.
2. Use the chamber control software to move the fiber to the chamber containing the pre-activating solution and incubate there for 3 min.
   Note: The pre-activating solution is weakly buffered for Ca²⁺, resulting in a very rapid activation and force development upon introduction of the fiber to the activating solution.
3. With 10 sec remaining in the pre-activating solution and the fiber length maintained at L_f, establish a zero force level in the experimental record.
   Note: The ‘Find Force Zero’ movement of the length-controller reveals the force-transducer level that corresponds to zero force (i.e. the fiber briefly becomes slack as a result of the movement). Passive force is the difference between that zero and the force level just prior to the transducer-zeroing movement.
4. At the end of the 3 min, 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.
5. After reaching maximum isometric force, use the length-controller to identify the force-transducer output that corresponds to zero force in the chamber containing the activating solution.
   Note: This is necessary because the force-transducer output that corresponds to zero force is, in general, different for each solution-filled chamber.
6. Following attainment of a second force plateau, return the fiber to the chamber containing the relaxing solution. Testing is now complete. To test multiple fibers during any one session aspirate all solutions and add new, chilled solutions.
   Note: Brenner’s cycling protocols should be considered when eliciting maximal contractions over an extended period of time. This protocol has been shown to conserve structural and mechanical properties in the maximally activated fiber⁹.