Evert L. de Beer

Doxorubicin and mechanical performance of cardiac trabeculae after acute and chronic treatment: a review

Doxorubicin, a very potent and often used anti-cancer drug, has a wide spectrum of biological activity. Classic studies have demonstrated that doxorubicin and other members of the anthracycline family intercalate with DNA and partially uncoil the double-stranded helix. Doxorubicin has a high affinity for cell nuclei: as much as 60% of the total intracellular amount of doxorubicin is found in the nucleus. Once binding to DNA occurs, several consequences may ensue. The binding of anthracyclines to DNA inhibits DNA polymerase and nucleic acid synthesis. In addition, anthracyclines are known to stabilize the otherwise cleavable complex between DNA and homodimeric topoisomerase II enzyme subunits, resulting in the formation of protein-linked DNA double strand breaks. In tumor cells, these anthracycline-induced perturbations are believed to result in a final common pathway of endonucleolytic DNA fragmentation known as apoptosis. Because proliferation is an important determinant of tumor growth, interference with the genome is regarded as the primary cause of the anti-tumor action of doxorubicin. Intercalation with DNA may not be important in the cardiotoxicity associated with doxorubicin therapy (see next section), because cardiac cell proliferation in humans stops after 2 months of age. This review is focussed on the effects of doxorubicin on mechanical performance in skinned cardiac trabeculae after acute and chronic administration of doxorubicin. We look especially at the mechanical performance and the molecular changes observed and related to mechanical performance.

Direct action of estradiol-17β on the atrial action potential

The effect of the two C-17 isomers of estradiol on the shape of the action potential of rat atrial tissue was studied by means of classical glass electrodes for different concentrations of estradiol. Resting potential and upstroke were not affected by estradiol, but the duration of the action potential was reduced. Only estradiol-17 beta exhibits an effect in a concentration dependent way, while estradiol-17 alpha has no effect at all. The ionic mechanism was studied by adding specific ionic blockers to the perfusate. Since the effect was much less pronounced when a slow inward current blocker was added, it was concluded that estradiol-17 beta acts mainly via the slow inward current channel. Only a small part of the interaction takes place via the potassium outward channel.

Doxorubicin interacts directly with skinned single skeletal muscle fibres

The effect of doxorubicin, a highly effective anticancer agent, on the contractile apparatus of skinned single muscle fibres was tested in a concentration of 1 microM. Sarcomere length was set and held at 2 microns. Doxorubicin induced an increase in tension dependent on the Ca2+ concentration and time of incubation. The rise was up to 25% at [Ca2+] 40 microM. A parallel, small but significant shift of the calcium sensitivity curve, the relation between normalized tension and the negative logarithm of [Ca2+], the pCa, was observed. The results of this study suggest a direct interaction of doxorubicin with the actin myosin structure, possibly by an effect on myosin-ATP activity.

Dexrazoxane pre-treatment protects skinned rat cardiac trabeculae against delayed doxorubicin-induced impairment of crossbridge kinetics.

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. 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−1 (i.v.), or were pre‐injected with DXR (40 mg kg−1, 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. Crossbridge turnover kinetics were studied after application of rapid length perturbations of varying amplitudes in Ca2+‐activated preparations. DXR treatment offered a significant protection against the DOX‐induced impairment of the crossbridge kinetics in isolated cardiac trabeculae. Time constants describing transitions between different crossbridge states were restored to normal in both the quick release protocol and the slack‐test. DXR prevented the shift from the ‘high ATPase’ α‐myosin heavy chain (MHC) isoform towards the ‘low‐ATPase’ β‐MHC isoform in the ventricles. We conclude that pre‐administration of DXR in rats greatly reduces the deleterious effects of chronic DOX treatment on the trabecular actin – myosin crossbridge cycle. Preventing direct deleterious effects 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.

The effect of length on the sensitivity to phenylephrine and calcium in intact and skinned vascular smooth muscle.

The length dependency of the sensitivity to activators of the smooth muscle of different blood vessels is not yet fully understood. Muscle preparations of the aorta, the femoral artery and the portal vein of the rabbit were investigated for the length dependency of the sensitivity to phenylephrine and calcium in both intact and triton X- 100 skinned preparations. For intact smooth muscles we found that at increased preparation length, the sensitivity of contraction was increased. The femoral artery showed the largest effect and the portal vein the smallest. In the skinned preparations of the three preparations the calcium sensitivity was not dependent on the preparation length. We conclude that the changes of the sensitivity in intact preparations are not caused by changes of the calcium sensitivity of the contractile proteins.

Ca2+ channel antagonists enhance tension in skinned skeletal and heart muscle fibres.

Striated muscle fibres, both skeletal and cardiac of different species including human, skinned by freeze-drying, were activated in solutions strongly buffered for Ca2+. The single fibres were immersed in solutions with different [Ca2+]. Sarcomere length was set and controlled by laser diffraction. Fibre type was determined by Sr2+ activation. The relation between the negative logarithm of the Ca2+ concentration and the normalized tension, the Ca2+ sensitivity curve, was investigated. The effect on the contractile machinery of three different Ca2+ channel antagonists (verapamil, diltiazem and nifedipine) in a therapeutic concentration (10(-6) M) was investigated. The possible effects on the Ca2+ sensitivity curve were quantified by: (1) the change in maximal tension developed at pCa2+ = 4.4; (2) the change in pCa2+ value at which 50% of the tension induced at pCa2+ = 4.4; (3) the steepness of the Ca2+ sensitivity curve in this point. The three drugs tested, at a therapeutic concentration of 1 microM, all enhanced maximal induced tension by respectively 25, 20 and 7%. The sarcomere length dependency of the effect proved to be dependent upon the drug, but also slightly on fibre type (skeletal or cardiac), or on species. It is concluded that the drug influences the cooperativity of the two different types of binding sites on troponin-C (low- and high-affinity sites). Tension enhancement was due to increased stiffness of the actin-myosin interaction site.

A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres.

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 S 1 during the transition.

Strain dependence of the elastic properties of force-producing cross-bridges in rigor skeletal muscle

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 Youngs modulus of 20% upon a stretch of 1 nm/half-sarcomere. Remarkably, the strain dependence of Youngs 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 Youngs modulus in rigor increases toward higher frequencies.

The effect of some sex hormones on the isovolumetric pressure curve of the rabbit's left ventricle

The effect of some sex hormones (estradiol-17 alpha), estradiol-17 beta, estrone, progesterone and testosterone) on the mechanical activity of rabbit left ventricle was studied in concentrations within the physiological range. Investigations were carried out during the first ten minutes after the introduction of the steroid. Only progesterone and estradiol-17 beta affect the isovolumetric pressure curve; the other steroids do not exhibit any effect. The effects of estradiol-17 beta and progesterone counterbalance each other. Estradiol-17 beta enhances the rate of pressure development and decreases the time to reach peak pressure. Progesterone decreases both the rate of pressure development and the time to reach peak pressure.

Immediate effects of diastolic loading variations on the left ventricular inotropic state in open chest dogs

The influence of left ventricular filling and variations in end diastolic volume on cardiac performance was studied in the intact dog heart. Left ventricular filling volumes and stroke volumes were calculated on a beat to beat basis from measurements of natural mitral inflow and aortic outflow obtained by electromagnetic flow sensors. Instantaneous controlled modifications of end diastolic volume were performed through a cannula situated in the left ventricle and connected to a pump system outside the dog. This system enabled controlled increases or decreases of the end diastolic volume at any prechosen moment during the diastolic pause. Absolute volume variations in end diastolic volume and end systolic volumes could be calculated by combining the integrated flow signals from different consecutive beats. Left ventricular performance was evaluated in terms of end systolic volume and end systolic pressure variations. When the diastolic volume was abruptly increased by the pump system, natural mitral inflow decreased but end diastolic volume increased. The effect on diastolic pressure was dependent on the variation in filling rate, the amplitude of the infusion, the moment at which the infusion was started, and the diastolic pressure at the start of the infusion. Also stroke volume, maximal systolic pressure (Pmax), end systolic pressure, and end diastolic volume increased. The increased systolic performance was attributed to the increased end diastolic volume as expected according to Starlings law. When end diastolic volume was rapidly decreased during diastole by the pump, natural filling volumes increased to compensate for the volume loss by the pump. End diastolic volume was, however, smaller indicating that full compensation was not achieved. Evaluation of ventricular performance in terms of end systolic pressure and end systolic volume showed a decreased end systolic pressure and increased end systolic volume compared with the control values. The effect of a pump withdrawal was 1.62(0.38) times larger than could be explained on the basis of Starlings law. After the infusion of adrenaline the intrinsic depression disappeared and the influence of the volume withdrawal on cardiac performance was as expected from the Starling mechanism.