Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity.

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.

The stiffness of rabbit skeletal actomyosin cross-bridges determined with an optical tweezers transducer.

Muscle contraction is brought about by the cyclical interaction of myosin with actin coupled to the breakdown of ATP. The current view of the mechanism is that the bound actomyosin complex (or "cross-bridge") produces force and movement by a change in conformation. This process is known as the "working stroke." We have measured the stiffness and working stroke of a single cross-bridge (kappa xb, dxb, respectively) with an optical tweezers transducer. Measurements were made with the "three bead" geometry devised by Finer et al. (1994), in which two beads, supported in optical traps, are used to hold an actin filament in the vicinity of a myosin molecule, which is immobilized on the surface of a third bead. The movements and forces produced by actomyosin interactions were measured by detecting the position of both trapped beads. We measured, and corrected for, series compliance in the system, which otherwise introduces large errors. First, we used video image analysis to measure the long-range, force-extension property of the actin-to-bead connection (kappa con), which is the main source of "end compliance." We found that force-extension diagrams were nonlinear and rather variable between preparations, i.e., end compliance depended not only upon the starting tension, but also upon the F-actin-bead pair used. Second, we measured kappa xb and kappa con during a single cross-bridge attachment by driving one optical tweezer with a sinusoidal oscillation while measuring the position of both beads. In this way, the bead held in the driven optical tweezer applied force to the cross-bridge, and the motion of the other bead measured cross-bridge movement. Under our experimental conditions (at approximately 2 pN of pretension), connection stiffness (kappa con) was 0.26 +/- 0.16 pN nm-1. We found that rabbit heavy meromyosin produced a working stroke of 5.5 nm, and cross-bridge stiffness (kappa xb) was 0.69 +/- 0.47 pN nm-1.

Basis of passive tension and stiffness in isolated rabbit myofibrils.

By examining the mechanical properties of isolated skeletal and cardiac myofibrils in calcium-free, ATP-containing solution, we attempted to separate the stiffness contribution of titin filaments from that of weakly bound cross bridges. Efforts to enhance weak cross-bridge binding by lowering ionic strength were met by clear contractile responses. Even at low temperature, myofibrils bathed in low-ionic-strength relaxing solution generated increased force and exhibited sarcomere shortening, apparently caused by active contraction. At normal ionic strength, myofibril stiffness, estimated from the force response to rapid sinusoidal oscillations, increased steadily with sarcomere extension up to a strain limit. No obvious stiffness contribution from weak cross bridges was detectable. Instead, the stiffness response, which was frequency dependent at all sarcomere lengths, was apparently generated by the viscoelastic titin filaments. During imposed stretch-hold ramps, both peak force/stiffness and the amount of subsequent stress relaxation increased with higher stretch rates, larger stretch amplitudes, and longer sarcomere lengths. We conclude that, for a truly relaxed myofibril, both passive force and dynamic stiffness principally reflect the intrinsic viscoelastic properties of the titin filaments.

Limits of titin extension in single cardiac myofibrils.

Passive force and dynamic stiffness were measured in relaxed, single myofibrils from rabbit ventricle over a wide range of sarcomere lengths, from approximately 2-5 microns. Myofibril stretch up to sarcomere lengths of approximately 3 microns resulted in a steady increase in both force and stiffness. The shape of the length-force and the length-stiffness curves remained fully reproducible for repeated extensions to a sarcomere length of approximately 2.7 microns. Above this length, myofibrillar viscoelastic properties were apparently changed irreversibly, likely due to structural alterations within the titin (connectin) filaments. Stretch beyond approximately 3 microns sarcomere length resulted in a markedly reduced slope of the passive force curve, while the stiffness curve became flat. Thus, cardiac sarcomeres apparently reach a strain limit near a length of 3 microns. Above the strain limit, both curve types frequently showed a series of inflections, which we assumed to result from the disruption of titin-thick filament bonds and consequent addition of previously bound A-band titin segments to the elastic I-band titin portion. Indeed, we confirmed in immunofluorescence microscopic studies, using a monoclonal antibody against titin near the A/I junction, that upon sarcomere stretch beyond the strain limit length, the previously stationary antibody epitopes suddenly moved into the I-band, indicating A-band titin release. Altogether, the passive force/stiffness-length relation of cardiac myofibrils was qualitatively similar to, but quantitatively different from, that reported for skeletal myofibrils. From these results, we inferred that cardiac myofibrils have an approximately two times greater relative I-band titin extensibility than skeletal myofibrils. This could hint at differences in the maximum passive force-bearing capacity of titin filaments in the two muscle types.

An optical fiber transducer for single myofibril force measurement.

A force transducer has been developed for use in force measurement of skeletal muscle myofibrils. The transducer is suitable for measurement of passive and contractile forces in a range up to 200 micrograms, with 1 microgram resolution. It is based upon the operating principle of the deflection of an optical fiber of known compliance, sensed by the differential illumination of two phototransistors. Attractive features include ease of operation and specimen mounting, high bandwidth, adaptability for different force ranges, and simple and inexpensive construction.

Active tension generation in isolated skeletal myofibrils.

Single or double myofibrils isolated from rabbit psoas muscle were suspended between a fine needle and an optical force transducer. By using a photodiode array, the length of every sarcomere along the specimen could be measured. Relaxed specimens exhibited uniform sarcomere lengths and their passive length-tension curve was comparable to that of larger specimens. Most specimens could be activated and relaxed four to five times before active force levels began to decline; some specimens lasted for 10-15 activation cycles. Active tension (20-22 degrees C) was reproducible from contraction to contraction. The contractile response was dependent on initial sarcomere length. If initially activated at sarcomere lengths of > or = 2.7 microns, one group of sarcomeres usually shortened to sarcomere lengths of 1.8-2.0 microns, while the remaining sarcomeres were stretched to longer lengths. Myofibrils that were carefully activated at shorter initial sarcomere lengths usually contracted homogeneously. Both homogeneous and inhomogeneous contractions produced high levels of active tension. Calcium sensitivity was found to be comparable to that in larger preparations; myofibrils immersed in pCa 6.0 solution generated 30% of maximal tension, while pCa 5.5-4.5 resulted in full activation. Active tension at full overlap of thick and thin filaments ranged from 0.34 to 0.94 N mm-2 (mean of 0.59 N mm-2 +/- 0.13 SD. n = 65). Even allowing for a maximum of 20% nonmyofibrillar space in skinned or intact muscle fibres, the mean tension generated by isolated myofibrils per cross-sectional area is higher than by fibre preparations.

Spontaneous sarcomeric oscillations at intermediate activation levels in single isolated cardiac myofibrils.

Spontaneous oscillations observed in various heart muscle preparations are widely thought to be triggered by spontaneous release of Ca2+ from the sarcoplasmic reticulum (SR). Here, we report undamped propagated oscillations that occur in the absence of SR. In single cardiac myofibrils treated with Triton X-100 to remove SR and held isometrically, partial activation initiated periodic fluctuations of sarcomere length persisting up to 1 hour. Oscillation characteristics could be readily quantitated by virtue of the small size of the preparation. In an individual sarcomere, the oscillation cycle generally consisted of a slow shortening phase, followed by a phase of rapid lengthening. Oscillations usually propagated along the myofibril--frequently along the entire specimen--in a wavelike fashion (average velocity, 12.3 microns/s at 10 degrees C; Q10, approximately 1.3). The oscillation period was 2.30 and 1.72 seconds at 10 degrees and 20 degrees C, respectively, and was insensitive to stretch. The average oscillation amplitude, which was temperature independent, decreased with stretch from more than 20% of the mean sarcomere length at lengths below 2 microns to zero beyond a sarcomere length of 3 microns. Stiffening of the Z line by labeling with anti-alpha-actinin resulted in a dose-dependent decrease of oscillation amplitude, while the period was not affected. Tension oscillations could not be detected in single myofibrils but were frequently detectable in myofibril doublets, where the oscillation magnitude (approximately 1 microgram) was above the noise floor. Addition of 10 mumol/L ryanodine to the activating solution did not alter oscillation characteristics, as expected, since the oscillations are unrelated to SR calcium release. On the basis of our results, we consider a mechanism for the oscillations in which a length dependence of myofibrillar Ca2+ sensitivity and a dynamic Z-line structure are essential.