Single muscle fibers, typically 2 to 3 mm in length, were randomly
teased from small glycerinated bundles at 0°C by means of jeweler’s
forceps. One end of the fiber was glued onto a force gauge (AE801;
SensoNor, Horten, Norway) and the other end onto a length
driver (P-841.10; Physik Instruments, Waldbronn, Germany). The fiber
was viewed through an inverted microscope (Fluorovert; Leitz, Wetzlar,
Germany) at a magnification of ×400, and the sarcomere length of the
fiber was adjusted to 2.5 μm by means of a calibrated eyepiece
micrometer. The temperature of the fiber bath solution was maintained
by means of a peltier module (KSM-0617; Komatsu Electronics, Tokyo,
Japan) and a temperature sensor (AD 590; Analog Devices,
Norwood, MA), which provided a feedback signal for a
custom-built proportional-integral controller. A digital thermometer
(KM-330; Kane Instruments, Bedford, MA) was located
in the fiber bath solution to provide an independent and continued
read-out of temperature.
Fiber length was perturbed by a signal that was software-generated and
introduced to the fiber via D/A conversion and the length driver. The
form of the signal was pseudorandom binary noise (PRBN), which enabled
the calculation of stiffness and phase spectra with greater resolution
and in less time compared with the sinusoidal technique.
14 The amplitude of the length signal was typically 0.05% of the fiber
length.
Single fibers were incubated for approximately 5 minutes in the muscle
bath filled with relaxing solution. Activation of the fiber was
achieved by changing from the well with relaxing solution to one
containing activation solution. The Ca2+ concentration of activation solution was pCa 4.0, which ensured maximal
activation of the fiber. The temperature of the bath solution was set
at 15°C. When steady tension had been attained, fiber length was
perturbed with the PRBN signal. The length changes together with the
resulting force responses of the fiber were sampled by the control
computer via A/D conversion. Fast Fourier transforms of the length and
force data yielded the stiffness and phase values. The stiffness and
phase data were smoothed, using a three-point convolution procedure,
and displayed on a digital plotter (7225A; Hewlett Packard, Palo Alto,
CA). f min was evaluated from
the dynamic stiffness plots by noting the frequency at which stiffness
was at the minimum.