Impact of tropomyosin isoform composition on fast skeletal muscle thin filament regulation and force development.

Tropomyosin (Tm) plays a central role in the regulation of muscle contraction and is present in three main isoforms in skeletal and cardiac muscles. In the present work we studied the functional role of α- and ßTm on force development by modifying the isoform composition of rabbit psoas skeletal muscle myofibrils and of regulated thin filaments for in vitro motility measurements. Skeletal myofibril regulatory proteins were extracted (78%) and replaced (98%) with Tm isoforms as homogenous ααTm or ßßTm dimers and the functional effects were measured. Maximal Ca(2+) activated force was the same in ααTm versus ßßTm myofibrils, but ßßTm myofibrils showed a marked slowing of relaxation and an impairment of regulation under resting conditions compared to ααTm and controls. ßßTm myofibrils also showed a significantly shorter slack sarcomere length and a marked increase in resting tension. Both these mechanical features were almost completely abolished by 10 mM 2,3-butanedione 2-monoxime, suggesting the presence of a significant degree of Ca(2+)-independent cross-bridge formation in ßßTm myofibrils. Finally, in motility assay experiments in the absence of Ca(2+) (pCa 9.0), complete regulation of thin filaments required greater ßßTm versus ααTm concentrations, while at full activation (pCa 5.0) no effect was observed on maximal thin filament motility speed. We infer from these observations that high contents of ßßTm in skeletal muscle result in partial Ca(2+)-independent activation of thin filaments at rest, and longer-lasting and less complete tension relaxation following Ca(2+) removal.

New techniques in linear and non-linear laser optics in muscle research.

This review proposes a brief summary of two applications of lasers to muscle research. The first application (laser tweezers), is now a well-established technique in the field, adopted by several laboratories in the world and producing a constant stream of original data, fundamental for our improved understanding of muscle contraction at the level of detail that only single molecule measurements can provide. As an example of the power of this technique, here we focus on some recent results, revealing the performance of the working stroke in at least two distinct steps also in skeletal muscle myosin. A second laser-based technique described here is second-harmonic generation; the application of this technique to muscle research is very recent. We describe the main results obtained thus far in this area and the potentially remarkable impact that this technology may have in muscle research.

No direct effect of creatine phosphate on the cross-bridge cycle in cardiac myofibrils.

Creatine phosphate (CP) and creatine kinase (CK) are involved in the rapid resynthesis of ATP and thereby serve to stabilize ATP concentration and to maintain free ADP low inside cardiac muscle cells during contraction. Recently, it has been suggested from experiments in permeabilized multicellular preparations that CP/CK also regulate the kinetics of the actomyosin interaction (cross-bridge cycle) and may explain contractile dysfunction, for instance, during ischemia. However, the reported effects of CP/CK may be confounded by diffusion limitations in multicellular preparations in which inorganic phosphate (P(i)) and ADP may significantly accumulate during contraction. To test this hypothesis, we measured force production and the rates of force development (k (ACT) and k (TR)) in isolated cardiac myofibrils, in which rapid concentration changes of Ca(2+), CP/CK, and P(i) were imposed using a rapid perfusion change system. The results showed that CP/CK did not influence maximum force-generating capacity, whereas P(i) markedly reduced force and increased the rates of force development. No effects of CP/CK on the rates of force development were observed, consistent with the notion that CP/CK do not exert a direct effect on the actomyosin interaction.

Mechanism of cross-bridge detachment in isometric force relaxation of skeletal and cardiac myofibrils.

Skeletal and cardiac muscle relaxation is governed by the interplay between two macromolecular systems: (i) membrane bound Ca2+ transport proteins and (ii) sarcomeric proteins. Photolysis experiments in skinned muscle preparations and fast solution switching studies in single myofibrils offer means for isolating sarcomeric mechanisms of relaxation from those related to myoplasmic Ca2+ removal. Single myofibril experiments have recently shown that cross-bridge mechanics and detachment kinetics are the major determinants of the time course of relaxation. Full force decay in myofibrils occurs in two phases: a slow one followed by a rapid one. The latter is initiated by sarcomere 'give' and dominated by inter-sarcomere dynamics while the former occurs under nearly isometric conditions. Strong evidence has been found that the slow rate of force decay in myofibril relaxation reflects the rate at which cross-bridges leave force-generating states under isometric conditions. Dissection of chemo-mechanical transduction process in myofibrils indicates that both forward and backward transitions of cross-bridges from force-generating to non-force-generating states contribute to muscle relaxation.

Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils.

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kACT), and the rate of tension redevelopment following a quick release and restretch (kTR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kACT or kTR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force-generating mechanism.

Characterization of the cross-bridge force-generating step using inorganic phosphate and BDM in myofibrils from rabbit skeletal muscles.

The inhibitory effects of inorganic phosphate (P(i)) on isometric force in striated muscle suggest that in the ATPase reaction P(i) release is coupled to force generation. Whether P(i) release and the power stroke are synchronous events or force is generated by an isomerization of the quaternary complex of actomyosin and ATPase products (AM.ADP.P(i)) prior to the following release of P(i) is still controversial. Examination of the dependence of isometric force on [P(i)] in rabbit fast (psoas; 5-15 degrees C) and slow (soleus; 15-20 degrees C) myofibrils was used to test the two-step hypothesis of force generation and P(i) release. Hyperbolic fits of force-[P(i)] relations obtained in fast and slow myofibrils at 15 degrees C produced an apparent asymptote as [P(i)]-->infinity of 0.07 and 0.44 maximal isometric force (i.e. force in the absence of P(i)) in psoas and soleus myofibrils, respectively, with an apparent K(d) of 4.3 mM in both. In each muscle type, the force-[P(i)] relation was independent of temperature. However, 2,3-butanedione 2-monoxime (BDM) decreased the apparent asymptote of force in both muscle types, as expected from its inhibition of the force-generating isomerization. These data lend strong support to models of cross-bridge action in which force is produced by an isomerization of the AM.ADP.P(i) complex immediately preceding the P(i) release step.

The effect of inorganic phosphate on force generation in single myofibrils from rabbit skeletal muscle.

In striated muscle, force generation and phosphate (P(i)) release are closely related. Alterations in the [P(i)] bathing skinned fibers have been used to probe key transitions of the mechanochemical coupling. Accuracy in this kind of studies is reduced, however, by diffusional barriers. A new perfusion technique is used to study the effect of [P(i)] in single or very thin bundles (1-3 microM in diameter; 5 degrees C) of rabbit psoas myofibrils. With this technique, it is possible to rapidly jump [P(i)] during contraction and observe the transient and steady-state effects on force of both an increase and a decrease in [P(i)]. Steady-state isometric force decreases linearly with an increase in log[P(i)] in the range 500 microM to 10 mM (slope -0.4/decade). Between 5 and 200 microM P(i), the slope of the relation is smaller ( approximately -0.07/decade). The rate constant of force development (k(TR)) increases with an increase in [P(i)] over the same concentration range. After rapid jumps in [P(i)], the kinetics of both the force decrease with an increase in [P(i)] (k(Pi(+))) and the force increase with a decrease in [P(i)] (k(Pi(-))) were measured. As observed in skinned fibers with caged P(i), k(Pi(+)) is about three to four times higher than k(TR), strongly dependent on final [P(i)], and scarcely modulated by the activation level. Unexpectedly, the kinetics of force increase after jumps from high to low [P(i)] is slower: k(Pi(-)) is indistinguishable from k(TR) measured at the same [P(i)] and has the same calcium sensitivity.

Modulation by substrate concentration of maximal shortening velocity and isometric force in single myofibrils from frog and rabbit fast skeletal muscle.

1. The effects of magnesium adenosine triphosphate (MgATP; also referred to as 'substrate') concentration on maximal force and shortening velocity have been studied at 5 C in single and thin bundles of striated muscle myofibrils. The minute diameters of the preparations promote rapid diffusional equilibrium between the bathing medium and lattice space so that during contraction fine control of substrate and product concentrations is achieved. 2. Myofibrils from frog tibialis anterior and rabbit psoas fast skeletal muscles were activated maximally by rapidly (10 ms) exchanging a continuous flux of pCa 8.0 for one at pCa 4.75 at a range of substrate concentrations from 10 microM to 5 mM. At high substrate concentrations maximal isometric tension and shortening velocity of both frog and rabbit myofibrils were very close to those determined in whole fibre preparations from the same muscle types. 3. As in frog and rabbit skinned whole fibres, the maximal isometric force of the myofibril preparations decreases as MgATP concentration is increased. The maximal velocity of unloaded shortening (V0) depends hyperbolically on substrate concentration. V0 extrapolated to infinite MgATP (3.6 +/- 0.2 and 0.8 +/- 0.03 l0 s-1 in frog and rabbit myofibrils, respectively) is very close to that determined directly at high substrate concentration. The Km is 210 +/- 20 microM for frog tibialis anterior and 120 +/- 10 microM for rabbit psoas myofibrils, values about half those found in larger whole fibre preparations of the same muscle types. This implies that measurements in whole skinned fibres are perturbed by diffusional delays, even in the presence of MgATP regenerating systems. 4. In both frog and rabbit myofibrils, the Km for V0 is about one order of magnitude higher than the Km for myofibrillar MgATPase determined biochemically in the same experimental conditions. This confirms that the difference between the Km values for MgATPase and shortening velocity is a basic feature of the mechanism of chemomechanical transduction in muscle contraction.

ATPase and shortening rates in frog fast skeletal myofibrils by time-resolved measurements of protein-bound and free Pi.

Shortening and ATPase rates were measured in Ca2+-activated myofibrils from frog fast muscles in unloaded conditions at 4 degrees C. ATPase rates were determined using the phosphate-binding protein method (free phosphate) and quench flow (total phosphate). Shortening rates at near zero load (V0) were estimated by quenching reaction mixtures 50 ms to 10 s old at pH 3.5 and measuring sarcomere lengths under the optical microscope. As with the rabbit psoas myofibrils (C. Lionne, F. Travers, and T. Barman, 1996, Biophys. J. 70:887-895), the ATPase progress curves had three phases: a transient Pi burst, a fast linear phase (kF), and a deceleration to a slow phase (kS). Evidence is given that kF is the ATPase rate of shortening myofibrils. V0 is in good agreement with mechanical measurements in myofibrils and fibers. Under the same conditions and at saturation in ATP, V0 and kF are 2.4 microm half-sarcomere(-1) s(-1) and 4.6 s(-1), and their Km values are 33 and 200 microM, respectively. These parameters are higher than found with rabbit psoas myofibrils. The myofibrillar kF is higher than the fiber ATPase rates obtained previously in frog fast muscles but considerably lower than obtained in skinned fibers by the phosphate-binding protein method (Z. H. He, R. K. Chillingworth, M. Brune, J. E. T. Corrie, D. R. Trentham, M. R. Webb, and M. R. Ferenczi, 1997, J. Physiol. 50:125-148). We show that, with frog as with rabbit myofibrillar ATPase, phosphate release is the rate-limiting step.

Force regulation by Ca2+ in skinned single cardiac myocytes of frog.

Atrial and ventricular myocytes 200 to 300 microm long containing one to five myofibrils are isolated from frog hearts. After a cell is caught and held between two suction micropipettes the surface membrane is destroyed by briefly jetting relaxing solution containing 0.05% Triton X-100 on it from a third micropipette. Jetting buffered Ca2+ from other pipettes produces sustained contractions that relax completely on cessation. The pCa/force relationship is determined at 20 degrees C by perfusing a closely spaced sequence of pCa concentrations (pCa = -log[Ca2+]) past the skinned myocyte. At each step in the pCa series quick release of the myocyte length defines the tension baseline and quick restretch allows the kinetics of the return to steady tension to be observed. The pCa/force data fit to the Hill equation for atrial and ventricular myocytes yield, respectively, a pK (curve midpoint) of 5.86 +/- 0.03 (mean +/- SE.; n = 7) and 5.87 +/- 0.02 (n = 18) and an nH (slope) of 4.3 +/- 0.34 and 5.1 +/- 0.35. These slopes are about double those reported previously, suggesting that the cooperativity of Ca2+ activation in frog cardiac myofibrils is as strong as in fast skeletal muscle. The shape of the pCa/force relationship differs from that usually reported for skeletal muscle in that it closely follows the ideal fitted Hill plot with a single slope while that of skeletal muscle appears steeper in the lower than in the upper half. The rate of tension redevelopment following release restretch protocol increases with Ca2+ >10-fold and continues to rise after Ca2+ activated tension saturates. This finding provides support for a strong kinetic mechanism of force regulation by Ca2+ in frog cardiac muscle, at variance with previous reports on mammalian heart muscle. The maximum rate of tension redevelopment following restretch is approximately twofold faster for atrial than for ventricular myocytes, in accord with the idea that the intrinsic speed of the contractile proteins is faster in atrial than in ventricular myocardium.

Calcium dependence of the apparent rate of force generation in single striated muscle myofibrils activated by rapid solution changes.

Single myofibrils or small groups of myofibrils were isolated from different types of striated muscle: rabbit psoas, frog tibialis anterior, frog atrial and ventricular muscle. The Ca2+ concentration of the solution perfusing the myofibrils was changed within few milliseconds by translating the interface between two flowing streams of solution across the preparations. In all types of myofibrils tested, the time course of force rise in response to maximal activation (pCa 4.75) was approximately monoexponential and nearly superimposable on that observed after a release-restretch protocol applied to the myofibril at the plateau of maximal contractions. This suggests that the kinetics of force development following rapid myofibril activation essentially reflects the kinetics of interaction between contractile proteins. The half time of force rise in response to maximal activation varied among different myofibril types; it was shortest in frog tibialis anterior myofibrils and longest in frog ventricular myofibrils. In all types of myofibril preparations tested the half time of force rise increased with decreasing Ca2+ levels in the activating solution. The finding provides support for a kinetic mechanism of force regulation by Ca2+ in all types of striated muscle. The extent of this Ca2+ effect, however, varied among the different myofibril preparations tested; at 15 degrees C for instance, it was smaller in frog tibialis anterior myofibrils than in the other preparations.

Active and passive forces of isolated myofibrils from cardiac and fast skeletal muscle of the frog.

1. Force measurements in isolated myofibrils (15 degrees C; sarcomere length, 2.10 microns) were used in this study to determine whether sarcomeric proteins are responsible for the large differences in the amounts of active and passive tension of cardiac versus skeletal muscle. Single myofibrils and bundles of two to four myofibrils were prepared from glycerinated tibialis anterior and sartorius muscles of the frog. Skinned frog atrial myocytes were used as a model for cardiac myofibrils. 2. Electron microscope analysis of the preparations showed that: (i) frog atrial myocytes contained a small and variable number of individual myofibrils (from 1 to 7); (ii) the mean cross-sectional area and mean number of myosin filaments of individual cardiac myofibrils did not differ significantly from those of single skeletal myofibrils; and (iii) the total myofibril cross-sectional area of atrial myocytes was on average comparable to that of bundles of two to four skeletal myofibrils. 3. In maximally activated skeletal preparations, values of active force ranged from 0.45 +/- 0.03 microN for the single myofibrils (mean +/- S.E.M.; n = 16) to 1.44 +/- 0.24 microN for the bundles of two to four myofibrils (n = 9). Maximum active force values of forty-five cardiac myocytes averaged 1.47 +/- 0.10 microN and exhibited a non-continuous distribution with peaks at intervals of about 0.5 microN. The results suggest that variation in active force among cardiac preparations mainly reflects variability in the number of myofibrils inside the myocytes and that individual cardiac myofibrils develop the same average amount of force as single skeletal myofibrils. 4. The mean sarcomere length-resting force relation of atrial myocytes could be superimposed on that of bundles of two to four skeletal myofibrils. This suggests that, for any given amount of strain, individual cardiac and skeletal sarcomeres bear essentially the same passive force. 5. The length-passive tension data of all preparations could be fitted by an exponential equation. Equation parameters obtained for both types of myofibrils were in reasonable agreement with those reported for larger preparations of frog skeletal muscle but were very different from those estimated for multicellular frog atrial preparations. It is concluded that myofibrils are the major determinant of resting tension in skeletal muscle; structures other than the myofibrils are responsible for the high passive stiffness of frog cardiac muscle.