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Stretch and release experiments carried out on skinned single fibers of frog skeletal muscle under rigor conditions indicate that the elastic properties of the fiber depend on strain. For modulation frequencies below 1000 Hz, the results show an increase in Young's modulus of 20% upon a stretch of 1 nm/half-sarcomere. Remarkably, the strain dependence of Young's modulus decreases at higher frequencies to about 10% upon a 1-nm/half-sarcomere stretch at a modulation frequency of 10 kHz. This suggests that the cause of the effect is less straightforward than originally believed: a simple slackening of the filaments would result in an equally large strain dependence at all frequencies, whereas strain-dependent properties of the actin filaments should show up most clearly at higher frequencies. We believe that the reduction of the strain dependence points to transitions of the cross-bridges between distinct force-producing states. This is consistent with the earlier observation that Young's modulus in rigor increases toward higher frequencies.

Monte Carlo simulations were employed to model FRET in systems that included interactions between multiple donors and acceptors and photobleaching. Simulations revealed that pixel-to-pixel variations in microscopy experiments can be due both to variations in the probe distribution and to photon noise, depending on the intensity and probes per detection pixel. The Monte Carlo simulations were used to describe fluorescence experiments on (single) actin filaments using a conventional fluorescence microscope. The filaments were labeled with TRITC at phalloidin and Cy5-maleimide at cys374 and the donor−acceptor distances were taken from literature. The images show a large pixel-to-pixel variation in the fluorescence intensity and suffer from photobleaching. In experiments, the main source of noise was the probe distribution. The acceptor suffered from rapid photobleaching, which resulted in an increase of the donor intensity in time and hampers the straightforward determination of the energy transfer efficiency. The photobleaching of both the donor and the acceptor have been included in the simulations, enabling accurate modeling of the time course of the fluorescence intensities of both donor and acceptor and the energy transfer efficiency.

1 Dexrazoxane (DXR, ICRF-187) has been shown both in animal studies and clinical trials to provide a substantial cardioprotection when co-administered with anthracycline drugs like Doxorubicin (DOX). In a previous study, we showed that chronic DOX treatment in rats is associated with a clear impairment of the crossbridge kinetics and shift in myosin iso-enzymes. 2 The present study was adopted to investigate whether the cardioprotective action of DXR involves preservation of the normal actin-myosin interaction. Rats were treated for 4 weeks with either DOX at a weekly dose of 2 mg kg 71 (i.v.), or were pre-injected with DXR (40 mg kg 71, i.v.) at a 20 : 1 dose ratio 30 min prior to the DOX infusion. Rats receiving saline or DXR alone were included in the experiments. Cardiac trabeculae were isolated 4 weeks after the last infusion and were skinned with detergent. 3 Crossbridge turnover kinetics were studied after application of rapid length perturbations of varying amplitudes in Ca 2+ -activated preparations. DXR treatment oered a signi®cant protection against the DOX-induced impairment of the crossbridge kinetics in isolated cardiac trabeculae. Time constants describing transitions between dierent crossbridge states were restored to normal in both the quick release protocol and the slack-test. DXR prevented the shift from the`high ATPase' amyosin heavy chain (MHC) isoform towards the`low-ATPase' b-MHC isoform in the ventricles. 4 We conclude that pre-administration of DXR in rats greatly reduces the deleterious eects of chronic DOX treatment on the trabecular actin ± myosin crossbridge cycle. Preventing direct deleterious eects on the actin ± myosin crossbridge system may provide a new target for preventing or reducing DOX-related cardiotoxicity and may enable patients to continue the treatment beyond currently imposed limits.

In this paper a versatile, high sensitivity spectrograph is presented for use in uorescence m icroscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-ofthe-art back illuminated charge-coupled device (CCD) camera. The spectrograph, including the CCD camera, has a detection ef ciency of 0.77 6 0.05 at 633 nm. Full emission spectra with a 1-5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. Two applications are shown, in which the spectrograph is ber-coupled to a commercia l confocal laser scanning microscope. In the rst example, Fö rster resonance energy transfer imaging experiments were carried out on double-labeled actin laments in the in vitro motility assay. A 160 3 160 point im age was recorded in 1.5 m in at 3 ms dwell time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S1 during the transition.

Freeze-dried skinned cardiac and skeletal muscle preparations of the rabbit were immersed in Ca2+-containing solutions with different concentrations of caffeine. The relation between the negative logarithm of the Ca2+ concentration (pCa) and normalized developed force was studied. The exact position of these Ca2+-sensitivity functions proved to be dependent on both the sarcomere length (monitored by means of laser diffraction) and caffeine concentration. High concentrations of caffeine induce a reversible fall in tension, particularly at low binding site saturation (low pCa) and long sarcomere lengths. At a concentration of 10 mM caffeine, the sarcomere length dependency of the Ca2+-sensitivity curves is markedly reduced for the rising part of the curve. Only the depressive effect of caffeine at high pCa remains. A possible mechanism of caffeine action is discussed.

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