Filament interaction in intact muscle fibers monitored by light scattering.

Abstract

Measurement of changes in optical properties of intact muscle fibers during contraction has proven to be difficult or, in some cases, impossible due to movement of the muscle relative to the incident beam. In this paper we describe a technique for immobilizing single fibers in clear gelatin, which permits measurement of light scattering signals undistorted by movement artifacts. We also describe the phase and amplitude relationship between changes in intensity of light scattering (at 900 to incident beam) and tensions induced by electrically activating single fibers. With tensions that range up to 50% P0 (P0 = maximal tension measured by exposure of fibers to 200 mM K+), the maximal increase in light scattering is about 25% of that for resting fibers. The scattering increase precedes tension, and at low temperatures the interval between the two peaks can be 50-100 msec. We interpret these data on intact fibers, as we did our earlier data from studies on skinned fibers, as indicating that increases in light scattering power of muscle are primarily due to attachment of myosin cross-bridges to actin filaments.In a recent study on chemically skinned rabbit psoas fibers (1), we found a large increase in the light scattering power of the fibers (900 to incident beam) upon induction of rigor and Ca2+ activation. The rigor-induced increase in scattering is maximal when thick-thin filament overlap is maximal and is nearly abolished by stretching fibers to lengths where overlap is minimal. This observation, as well as other data, led us to propose that the increase in light scattering accompanying rigor and contraction results from the attachment of myosin crossbridges to actin filaments.In the course of extending the study to include contracting intact fibers we found, as did others (2,3), that movement of the fibers during development of tension distorts the optical signal. We have, in large measure, eliminated movement artifacts by embedding fibers in gelatin. This method and its validation are described in the text. In our earlier study on skinned fibers (1) only steady-state signals were measured. With the high reproducibility of these signals and with no apparent effect of fiber movement, immobilization by gelatin was not necessary.The present findings on intact fibers are in accord with our previous data on skinned fibers-i.e., a large increase in light scattering accompanies thick-thin filament interaction. The data further disclose that the increase in scattering precedes tension, and the magnitude of the lead time is temperature dependent. According to current cross-bridge models (4) and biochemical schemes of the ATP hydrolysis cycle (5) states involving cross-bridge attachment lead the force-generating step.Of relevance to the interpretation of our data on intact crayfish fibers are recent studies relating the time course of tension in frog fibers to cross-bridge extension as measured by low angle x-ray scattering (6) and myoplasmic Ca2+ concentration variations measured by aequorin luminescence (7). M...

Excitation-contraction coupling: role of K-activation within the transverse tubular system.

“…Standard electrophysiological techniques using current and voltage microelectrodes (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) (Fig. 1) confirmed earlier observations on whole-muscle (15) and fiber-bundle (14) preparations.…”

“…500 msec) Ca spikes with twitch tensions that approach 30% P0 (see figure 5 in ref. 20). However, crustacean fibers depolarized to similar potentials for 500 msec in the absence of Et4N generate tensions very close to P0 (6,29).…”

“…However, in fibers soaked for [15][16][17][18][19][20] hr in 50-100 mM Et4N, Ca spikes and tensions with comparable amplitudes and time courses to those shown in Fig. 1 were obtained.…”

Calcium accumulation by the sarcoplasmic reticulum in two populations of chemically skinned human muscle fibers. Effects of calcium and cyclic AMP.

“…Ca Uptake in the Presence of Oxalate In the presence of oxalate, active transport of Ca leads to formation of Ca oxalate crystals in the lumen of the SR (Hasselbach and Makinose, 1961) and to a progressive increase in light scattering by the fiber that is proportional to the increase in Ca content (Sorenson et al, 1980) . The experimental setup was slightly modified from that described previously , with a photodiode replacing the photomultiplier as detector of the scattered light (Katz et al, 1979) . The reaction chamber was mounted on the stage of an inverted microscope and the light scattered by the fiber was packed up from below through a long-working distance, 20X objective focused on the fiber.…”

Beer–Lambert law for optical tissue diagnostics: current state of the art and the main limitations

“…5 The impacts of absorption and scattering can be measured separately by time-resolved spectroscopy or intensity-modulated spectroscopy, therefore the scattering cross-talk can be estimated and BLL still be used. One should note, however, that light scattering in the tissues changes during muscle activation due to tension kinetics 32 and during neuronal activation in the brain under physiological or pathophysiological conditions. 1,33 Sometimes scattering can be assumed to be wavelength-independent if an algorithm with wavelength-dependent OPL is applied.…”