Measuring the Relationship between Force, Energy Consumption and Calcium Concentrations in Muscle Fibers using WPI-SIH Research Systems

Muscle contraction and relaxation is caused by the attachment and detachment of two types of molecules (actin and myosin) to each other within the fibers that compose a muscle. These molecules are arranged as overlapping filaments within the functional units of the muscle fibers that are known as sarcomeres. Each crossbridge that is made between the end of a myosin molecule and a binding site on an actin molecule requires a molecule of ATP, an energy source, to be hydrolyzed when the end of the myosin molecule is released from the actin filament. After the release, the myosin molecule is ready to move down to another actin binding site causing the sarcomere, and the muscle, to shorten. When ATP is regenerated through a series of enzymatic reactions, another molecule, NADH, provides the energy needed for the regeneration of the ATP.

The NADH fluoresces when exposed to ultraviolet (UV) light. The oxidized form of the molecule NAD+, which has lost its stored energy, does not fluoresce. So, any reduction in the fluorescence of the NADH-containing solution that is surrounding the muscle preparation indicates an increase in the activity of the enzyme (ATPase) that releases the energy of the ATP molecule for use in muscle contraction. This technique can only be performed on skinned muscle fibers, which are fibers that have had their membranes removed. The removal of membranes from the muscle fibers permits the free movement of molecules between the cells and their incubation solutions.

In the WPI ATPase activity system, the skinned muscle fiber preparation is incubated in a series of cuvettes containing solutions with the enzymes and substrates needed for the reactions of muscle contraction. The fibers are illuminated with a beam of UV light to fluoresce the NADH in the incubation solution. As ATPase activity occurs, the decrease in the fluorescence of the incubation solution is measured by a photometer. Because the decrease in NADH fluorescence is proportional to the increase in ATPase activity, the slope of the fluorescence decay curve provides a measure of the ATPase activity and ATP consumption in the fiber in proportion to the amount of force that the fiber generates.

The WPI SI-BF-100 Biofluorometer, when configured for ATPase activity measurements, includes all the components needed to measure and record ATPase activity in skinned fibers. When combined with an SI-MKB  muscle tester, you can measure the force of contraction from muscle fibers at the same time.


Intracellular Calcium Concentrations in Intact Muscle Fibers

When muscles are stimulated by the neurotransmitters released from motor nerves, muscle fibers develop action potentials that spread into the depths of the muscle through a network of tubules. These action potentials alter the membranes inside the fibers and cause the release of calcium from organelles, known as the sarcoplasmic reticulum, that are inside the fibers. The calcium released from the sarcoplasmic reticulum is also required for the binding of the myosin and actin in the muscle fibers. Calcium binds to the actin filament and causes the binding site for myosin to open and be available for the end of the myosin molecule to bind to the actin. Then, the end of the myosin molecule pulls the actin filament toward the center of the sarcomere and the sarcomere shortens, as does the muscle fiber. If a deficiency in the concentration of calcium in the fluid around the muscle fibers is created, the myosin binding sites on the actin filaments are covered and the fiber contraction is stopped. 

The intracellular concentrations of calcium are determined by measuring the fluorescence of a calcium-indicator dye like FURA. The lipid-soluble form of FURA is easily loaded into intact muscle fibers, where the dye portion of the FURA molecule is released and free to bind to calcium. When the dye binds to calcium, it fluoresces. Since the dye is also trapped inside the fiber, any fluorescence that is measured indicates the amount of calcium inside the fiber.

The SI-BF-100 Biofluorometer, when configured for intracellular calcium concentration measurements, includes all the components needed to measure and record calcium concentration inside fibers. When combined with an SI-MKB  muscle tester, you can measure the force of contraction from muscle fibers at the same time. WPI’s SI-BF-100 Biofluorometer works with the SI-H line of muscle testers. It measures intracellular calcium using LEDs that emit light in the 340nm and 380nm spectra and a photomultiplier that captures and magnifies the emission signal. The unit also has a feedback mechanism to monitor the actual light output of the Biofluorometer and make required adjustments to the output readings.

 FURA-2 dye is an ion-sensitive indicator that emits light (fluoresces) when it is excited with a 340nm or 380nm light beam. FURA-1 fluorescence properties change when calcium binds to it. The FURA-2 dye attached to Ca2+ that is tightly bound fluoresces when it is excited with a 340nm light beam. Likewise, the dye attached to free Ca2+ fluoresces when excited with a 380nm light beam. By alternately exciting at the two wavelengths, we can measure the ratio of the fluorescence intensity and use it to determine the concentration ratio of the bound and free calcium.

If the fluorescence is measured at 510nm, the fluorescence emission intensity depends on the excitation wavelength. Each curve corresponds to the calcium concentration noted on the graph.

Fluorescence intensity

The higher the concentration of calcium bound to the FURA-2 dye, the greater the fluorescence when excited at 340nm.

At shorter exciting light wavelengths, the fluorescence decreases with decreasing calcium concentration. At longer exciting light wavelengths, the fluorescence intensity increases with decreasing calcium concentration.

Fluorescence intensity

The 340nm and 380nm wavelengths are highlighted to show typical responses of FURA-2 dye.

Historically, a light source, filter wheel and photomultiplier were used to excite the dye at the proper wavelength. Now, using LED modules, we have much greater control over the measurement cycle. You can set the length of time each LED fires and establish the firing sequence. In addition, you can define the intensity of the LED signals, determine the number of readings used to calculate the rolling average of the ratio displayed, and set the photomultiplier filter frequency.

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