Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.     

Thick filament movement and isometric tension in activated skeletal muscle.

Thick filaments can move from the center of the sarcomere to the Z-disc while the isometric tension remains stable in skinned rabbit psoas fibers activated for several minutes (Horowits and Podolsky, 1987). Using the active and resting tension-length relations and the force-velocity relation, we calculated the time course and mechanical consequences of thick filament movement in the presence and absence of the elastic titin filaments, which link the ends of the thick filaments to the Z-discs and give rise to the resting tension. The calculated time course of thick filament movement exhibits a lag phase, during which the velocity and extent of movement are extremely small. This lag phase is dependent only on the properties of the cross-bridges and the initial position of the thick filament. The time course of thick filament movement in skinned rabbit psoas fibers at 7 degrees C is well fit assuming a small initial thick filament displacement away from the center of the sarcomere; this leads to a lag of approximately 80 s before any significant thick filament movement occurs. In the model incorporating titin filaments, this lag is followed by a phase of slow, steady motion during which isometric tension is stable. The model excluding titin filaments predicts a phase of acceleration accompanied by a 50% decrease in tension. The observed time course of movement and tension are consistent with the model incorporating titin filaments. The long lag phase suggests that in vivo, significant movement of thick filaments is unlikely to occur during a single contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compliance of thin filaments in skinned fibers of rabbit skeletal muscle.

The mechanical compliance (reciprocal of stiffness) of thin filaments was estimated from the relative compliance of single, skinned muscle fibers in rigor at sarcomere lengths between 1.8 and 2.4 micron. The compliance of the fibers was calculated as the ratio of sarcomere length change to tension change during imposition of repetitive cycles of small stretches and releases. Fiber compliance decreased as the sarcomere length was decreased below 2.4 micron. The compliance of the thin filaments could be estimated from this decrement because in this range of lengths overlap between the thick and thin filaments is complete and all of the myosin heads bind to the thin filament in rigor. Thus, the compliance of the overlap region of the sarcomere is constant as length is changed and the decrease in fiber compliance is due to decrease of the nonoverlap length of the thin filaments (the I band). The compliance value obtained for the thin filaments implies that at 2.4-microns sarcomere length, the thin filaments contribute approximately 55% of the total sarcomere compliance. Considering that the sarcomeres are approximately 1.25-fold more compliant in active isometric contractions than in rigor, the thin filaments contribute approximately 44% to sarcomere compliance during isometric contraction.     

Elastic behavior of connectin filaments during thick filament movement in activated skeletal muscle

Connectin (also called titin) is a huge, striated muscle protein that binds to thick filaments and links them to the Z-disc. Using an mAb that binds to connectin in the I-band region of the molecule, we studied the behavior of connectin in both relaxed and activated skinned rabbit psoas fibers by immunoelectron microscopy. In relaxed fibers, antibody binding is visualized as two extra striations per sarcomere arranged symmetrically about the M-line. These striations move away from both the nearest Z-disc and the thick filaments when the sarcomere is stretched, confirming the elastic behavior of connectin within the I- band of relaxed sarcomeres as previously observed by several investigators. When the fiber is activated, thick filaments in sarcomeres shorter than 2.8 microns tend to move from the center to the side of the sarcomere. This translocation of thick filaments within the sarcomere is accompanied by movement of the antibody label in the same direction. In that half-sarcomere in which the thick filaments move away from the Z-disc, the spacings between the Z-disc and the antibody and between the antibody and the thick filaments both increase. Conversely, on the side of the sarcomere in which the thick filaments move nearer to the Z-line, these spacings decrease. Regardless of whether I-band spacing is varied by stretch of a relaxed sarcomere or by active sliding of thick filaments within a sarcomere of constant length, the spacings between the Z-line and the antibody and between the antibody and the thick filaments increase with I-band length identically. These results indicate that the connectin filaments remain bound to the thick filaments in active fibers, and that the elastic properties of connectin are unaltered by calcium ions and cross-bridge activity.     

Viscoelasticity of the sarcomere matrix of skeletal muscles. The titin-myosin composite filament is a dual-stage molecular spring.

The mechanical roles of sarcomere-associated cytoskeletal lattices were investigated by studying the resting tension-sarcomere length curves of mechanically skinned rabbit psoas muscle fibers over a wide range of sarcomere strain. Correlative immunoelectron microscopy of the elastic titin filaments of the endosarcomeric lattice revealed biphasic extensibility behaviors and provided a structural interpretation of the multiphasic tension-length curves. We propose that the reversible change of contour length of the extensible segment of titin between the Z line and the end of thick filaments underlies the exponential rise of resting tension. At and beyond an elastic limit near 3.8 microns, a portion of the anchored titin segment that adheres to thick filaments is released from the distal ends of thick filament. This increase in extensible length of titin results in a net length increase in the unstrained extensible segment, thereby lowering the stiffness of the fiber, lengthening the slack sarcomere length, and shifting the yield point in postyield sarcomeres. Thus, the titin-myosin composite filament behaves as a dual-stage molecular spring, consisting of an elastic connector segment for normal response and a longer latent segment that is recruited at and beyond the elastic limit of the sarcomere. Exosarcomeric intermediate filaments contribute to resting tension only above 4.5 microns. We conclude that the interlinked endo- and exosarcomeric lattices are both viscoelastic force-bearing elements. These distinct cytoskeletal lattices appear to operate over two ranges of sarcomere strains and collectively enable myofibrils to respond viscoelastically over a broad range of sarcomere and fiber lengths.     

Resting Sarcomere Length-Tension Relation in Living Frog Heart

The sarcomere pattern and tension of isolated resting frog atrial trabeculae were continuously monitored. In the absence of any resting tension the sarcomere lengths varied with the diameter of the trabeculae. In over 75 % of the trabeculae the value exceeded 2.05 µm, the estimated in vivo length of the thin filaments, and it was never less than 1.89 µm. When the trabeculae were stretched the increase in length of the central undamaged portion could be completely accounted for by an increase in sarcomere length. The width of the A band was constant only at sarcomere lengths between 2.3 and 2.6 µm it decreased at smaller and increased at larger sarcomere lengths. A group of spontaneously active cells stretched the sarcomeres in cells in series to longer lengths than could be produced by passive tension applied to the ends of the trabeculae, but they did not influence the sarcomeres of adjacent cells. It is proposed that the connective tissue is a major factor in determining sarcomere length and that there are interactions between thick and thin filaments in resting muscles.     

Passive tension and stiffness of vertebrate skeletal and insect flight muscles: the contribution of weak cross-bridges and elastic filaments.

Tension and dynamic stiffness of passive rabbit psoas, rabbit semitendinosus, and waterbug indirect flight muscles were investigated to study the contribution of weak-binding cross-bridges and elastic filaments (titin and minititin) to the passive mechanical behavior of these muscles. Experimentally, a functional dissection of the relative contribution of actomyosin cross-bridges and titin and minititin was achieved by 1) comparing mechanically skinned muscle fibers before and after selective removal of actin filaments with a noncalcium-requiring gelsolin fragment (FX-45), and 2) studying passive tension and stiffness as a function of sarcomere length, ionic strength, temperature, and the inhibitory effect of a carboxyl-terminal fragment of smooth muscle caldesmon. Our data show that weak bridges exist in both rabbit skeletal muscle and insect flight muscle at physiological ionic strength and room temperature. In rabbit psoas fibers, weak bridge stiffness appears to vary with both thin-thick filament overlap and with the magnitude of passive tension. Plots of passive tension versus passive stiffness are multiphasic and strikingly similar for these three muscles of distinct sarcomere proportions and elastic proteins. The tension-stiffness plot appears to be a powerful tool in discerning changes in the mechanical behavior of the elastic filaments. The stress-strain and stiffness-strain curves of all three muscles can be merged into one, by normalizing strain rate and strain amplitude of the extensible segment of titin and minititin, further supporting the segmental extension model of resting tension development.     

Stiffness of glycerinated rabbit psoas fibers in the rigor state. Filament-overlap relation

The stiffness of glycerinated rabbit psoas fibers in the rigor state was measured at various sarcomere lengths in order to determine the distribution of the sarcomere compliance between the cross-bridge and other structures. The stiffness was determined by measuring the tension increment at one end of a fiber segment while stretching the other end of the fiber. The contribution of the end compliance to the rigor segments was checked both by laser diffractometry of the sarcomere length change and by measuring the length dependence of the Young's modulus; the contribution was found to be small. The stiffness in the rigor state was constant at sarcomere lengths of 2.4 microns or less; at greater sarcomere lengths the stiffness, when corrected for the contribution of resting stiffness, scaled with the amount of overlap between the thick and thin filaments. These results suggest that the source of the sarcomere compliance of the rigor fiber at the full overlapping of filaments is mostly the cross-bridge compliance.     

Extraction of Thick Filaments in Individual Sarcomeres Affects Force Production by Single Myofibrils

It has been accepted that the force produced by a skeletal muscle myofibril depends on its cross-sectional area but not on the number of active sarcomeres because they are arranged in series. However, a previous study performed by our group showed that blocking actomyosin interactions within an activated myofibril and depleting the thick filaments in one sarcomere unexpectedly reduced force production. In this study, we examined in detail how consecutive depletion of thick filaments in individual sarcomeres within a myofibril affects force production. Myofibrils isolated from rabbit psoas were activated and relaxed using a perfusion system. An extra microperfusion needle filled with a high-ionic strength solution was used to erase thick filaments in individual sarcomeres in real time before myofibril activation. The isometric forces were measured upon activation. The force produced by myofibrils with intact sarcomeres was significantly higher than the force produced by myofibrils with one or more sarcomeres lacking thick filaments (p < 0.0001) irrespective of the number of contractions imposed on the myofibrils and their initial sarcomere length. Our results suggest that the myofibril force is affected by intersarcomere dynamics and the number of active sarcomeres in series.     

Lattice swelling with the selective digestion of elastic components in single-skinned fibers of frog muscle.

Changes in the 1.0 lattice spacing during trypsin (0.25 micrograms/ml) treatment in mechanically skinned single fibers of frog muscle was examined by an x-ray diffraction method at various sarcomere lengths. The resting tension of a relaxed fiber was decreased by trypsin treatment but the stiffness of a rigor fiber was not, suggesting that elastic components were selectively digested. With progression of the digestion, the lattice spacing increased remarkably at longer sarcomere lengths and finally became independent of the sarcomere length. The increase in the lattice spacing was proportional to the decrease in the resting tension. These results support our previous suggestion (Higuchi, H., and Y. Umazume, 1986, Biophys. J., 50:385-389) that the lattice spacing decreases at long lengths due to compressive force exerted by a lateral elastic component that connects thick filaments to an axial elastic component. Consequently, it is unlikely that the decrease in the lattice spacing is determined by a decrease in the repulsive force between thick and thin filaments with stretching a fiber.     

Ultrastructure of Developing Flight Muscle in Drosophila. I. Assembly of Myofibrils

In order to evaluate the effects of specific mutations on sarcomere assembly and function in vivo, we describe the course of normal development of Drosophila indirect flight muscle (IFM) in staged pupae using electron microscopy. We find that no contractile assemblies remain in larval muscle remnants invaded by imaginal myoblasts, establishing that myofibrils in IFM assemble de novo. Stress-fiber-like structures or other template structures are not prominent before or during sarcomere assembly. By 42 hr pupation (eclosion 112 hr), thick and thin filaments have appeared simultaneously in slender, interdigitated arrays between regularly spaced Z-bodies. Each tiny, uniformly striated myofibril forms within a "sleeve" of microtubules, and both microtubules and myofibrils are attached to the cell membrane at each end of the fiber from the initial stages of assembly. Later in pupation, the microtubule "sleeves" disassemble. Sarcomere number appears to remain constant. We saw no evidence that terminal sarcomeres are sites for addition of new sarcomeres or that Z-lines split transversely, producing new, very short sarcomeres. Rather, initial thick and thin filaments and sarcomeres are much shorter than adult length. Sarcomere length increases smoothly and coordinately from 1.7 to 3.2 μm, reflecting increase in filament lengths and indicating that myosin and actin molecules must be incorporated into filaments after sarcomere formation. Myofilaments are not seen scattered in the cytoplasm at any time, nor do we detect filaments that could be in the process of being "trolleyed" along myofibrils into positions of lateral register. Myofibril diameter increases uniformly from 4-thick filaments to 36-thick filaments across, by peripheral addition of myofilaments. At each successive stage, all sarcomeres in a fiber attained similar length and diameter. Initial thick filaments are solid but within several hours these and all subsequently assembled thick filaments appear hollow. Initial Z-bodies do not show any internal lattice and are more irregularly shaped than adult Z-discs.     

Chicken breast muscle connectin: passive tension and I-band region primary structure

We performed cDNA cloning of chicken breast muscle connectin. Together with previous results, our analysis elucidated a 24.2 kb sequence encoding the amino terminus of the protein. This corresponded to the I-band region of the skeletal muscle sarcomere, which is involved in extension and contraction between the Z-line and the A-I junction. There were fewer middle immunoglobulin domains and amino acid residues in the PEVK segment of chicken breast muscle connectin than in human skeletal muscle connectin, but more than in human cardiac muscle connectin. We measured passive tension generation by stretching mechanically skinned myofibril bundles. This revealed that appreciable tension development in chicken breast muscle began at longer sarcomere spacings than in rabbit cardiac muscle, but at shorter spacings than in rabbit psoas and soleus muscles. We suggest that the chicken breast muscle sarcomere remains in a relatively extended state even in unstrained sarcomeres. This would explain why chicken breast muscle does not extend under force to the same degree as rabbit psoas and soleus muscles.     

Electron Microscopic Studies of Skeletal and Cardiac Muscle of a Benthic Fish. I. Myofibrillar Structure in Resting and Contracted Muscle

SYNOPSIS. Electron microscopic studies are reported on glycerinatedskeletal and cardiac muscle of a benthic fish, Coryphaenoidesspecies. In white skeletal muscle, the sarcomeres have a restinglength of approximately 1.8 µ, with thick filaments 1.4µ and thin filaments 0.75 µ in length. These dimensionsare somewhat shorter than filament lengths of oilier vertebratemuscles, possibly due to the elfect of volume increase duringassembly of thick and thin filaments at high hydrostatic pressure.During ATP-induced contraction of Coryphaenoides muscle fromsarcomere lengths of 1.8 µ to 1.6 µ, there is acharacteristic interdigitation of thick and thin filaments,with decrease in I band length and no change in length of thickor thin filaments. However, in sarcomeres contracted to lengthsof 1.5 µ. to 1.2 µ, there is a slight shorteningof the A band, apparently due to shortening of thick filaments,that occurs despite the presence of residual I band in the samesarcomeres. There is no obvious crumpling or distortion of thickfilaments during contraction to sarcomere lengths as low as1.0 µ, but filament organization undergoes extensive disarrayat sarcomere lengths approaching 0.7 µ. Although effectsfrom heterogeneity of filament length cannot be excluded withcertainty, the present evidence does suggest that contractionot Coryphaenoides muscle from 1.6 µ to 1.0 µ sarcomerelengih is accompanied by shortening of thick filaments consequentto a structural change within the thick filament core.     

Overextended sarcomeres regain filament overlap following stretch

Sarcomere overextension has been widely implicated in stretch-induced muscle injury. Yet, sarcomere overextensions are typically inferred based on indirect evidence obtained in muscle and fibre preparations, where individual sarcomeres cannot be observed during dynamic contractions. Therefore, it remains unclear whether sarcomere overextensions are permanent following injury-inducing stretch-shortening cycles, and thus, if they can explain stretch-induced force loss. We tested the hypothesis that overextended sarcomeres can regain filament overlap in isolated myofibrils from rabbit psoas muscles. Maximally activated myofibrils (n=13) were stretched from an average sarcomere length of 2.6±0.04μm by 0.9μm sarcomere(-1) at a speed of 0.1μm sarcomere(-1)s(-1) and immediately returned to the starting lengths at the same speed (sarcomere strain=34.1±2.3%). Myofibrils were then allowed to contract isometrically at the starting lengths (2.6μm) for ～30s before relaxing. Force and individual sarcomere lengths were measured continuously. Out of the 182 sarcomeres, 35 sarcomeres were overextended at the peak of stretch, out of which 26 regained filament overlap in the shortening phase while 9 (～5%) remained overextended. About 35% of the sarcomeres with initial lengths on the descending limb of the force-length relationship and ～2% of the sarcomeres with shorter initial lengths were overextended. These findings provide first ever direct evidence that overextended sarcomeres can regain filament overlap in the shortening phase following stretch, and that the likelihood of overextension is higher for sarcomeres residing initially on the descending limb.     

The effect of sarcomere non-uniformity on the sarcomere length-tension relationship of skinned fibers

It has proved difficult to activate skinned muscle fibers to produce high tension (3 kg/cm² level) without loss of clear striations. A new method was developed which permits high tension production in skinned muscle fibers while retaining clear striations. Clear striations allow reliable measurement of the sarcomere lengths during contraction by microscopy and diffractometry. The method is to increase the Ca⁺⁺ concentration of the bathing solution very gradually over a time period of 5 to 10 minutes. Once the skinned fiber is conditioned by this slow activation, subsequent contractions can be elicited by ordinary quick activations without loss of striations. When the experiments are carried out with careful controls for the uniformity of the sarcomere length distribution along the entire length of the fiber, contractions are highly repeatable. Using the new method and stringent quality control of fibers, the sarcomere length-isometric tension relationship of skinned rabbit soleus fibers was obtained. The results differ from those previously obtained by conventional activation methods in that tension increases with sarcomere length not only at low (pCa = 5.8), but also at high (pCa = 5.2), calcium concentration.     

CONTRACTILITY AND ULTRASTRUCTURE IN GLYCEROL-EXTRACTED MUSCLE FIBERS : I. The Relationship of Contractility to Sarcomere Length

This study was undertaken to determine whether glycerol-extracted rabbit psoas muscle fibers can develop tension and shorten after being stretched to such a length that the primary and secondary filaments no longer overlap. A method was devised to measure the initial sarcomere length and the ATP-induced isotonic shortening in prestretched isolated fibers subjected to a small preload (0.02 to 0.15 P₀). At all degrees of stretch, the fiber was able to shorten (60 to 75 per cent): to a sarcomere length of 0.7 µ when the initial length was 3.7 µ or less, and to an increasing length of 0.9 to 1.8 µ with increasing initial sarcomere length (3.8 to 4.4 µ). At sarcomere lengths of 3.8 to 4.5 µ, overlap of filaments was lost, as verified by electron microscopy. The variation in sarcomere length within individual fibers has been assessed by both light and electron microscopic measurements. In fibers up to 10 mm in length the stretch was evenly distributed along the fiber, and with sarcomere spacings greater than 4 µ there was only a slight chance of finding sarcomeres with filament overlap. These observations are in apparent contradiction to the assumption that an overlap of A and I filaments is necessary for tension generation and shortening.     

Tropomodulin is associated with the free (pointed) ends of the thin filaments in rat skeletal muscle

The length and spatial organization of thin filaments in skeletal muscle sarcomeres are precisely maintained and are essential for efficient muscle contraction. While the major structural components of skeletal muscle sarcomeres have been well characterized, the mechanisms that regulate thin filament length and spatial organization are not well understood. Tropomodulin is a new, 40.6-kD tropomyosin-binding protein from the human erythrocyte membrane skeleton that binds to one end of erythrocyte tropomyosin and blocks head-to-tail association of tropomyosin molecules along actin filaments. Here we show that rat psoas skeletal muscle contains tropomodulin based on immunoreactivity, identical apparent mobility on SDS gels, and ability to bind muscle tropomyosin. Results from immunofluorescence labeling of isolated myofibrils at resting and stretched lengths using anti-erythrocyte tropomodulin antibodies indicate that tropomodulin is localized at or near the free (pointed) ends of the thin filaments; this localization is not dependent on the presence of myosin thick filaments. Immunoblotting of supernatants and pellets obtained after extraction of myosin from myofibrils also indicates that tropomodulin remains associated with the thin filaments. 1.2-1.6 copies of muscle tropomodulin are present per thin filament in myofibrils, supporting the possibility that one or two tropomodulin molecules may be associated with the two terminal tropomyosin molecules at the pointed end of each thin filament. Although a number of proteins are associated with the barbed ends of the thin filaments at the Z disc, tropomodulin is the first protein to be specifically located at or near the pointed ends of the thin filaments. We propose that tropomodulin may cap the tropomyosin polymers at the pointed end of the thin filament and play a role in regulating thin filament length.     

ULTRASTRUCTURE OF THE RESTING AND CONTRACTED STRIATED MUSCLE FIBER AT DIFFERENT DEGREES OF STRETCH

Passive stretch, isometric contraction, and shortening were studied in electron micrographs of striated, non-glycerinated frog muscle fibers. The artifacts due to the different steps of preparation were evaluated by comparing sarcomere length and fiber diameter before, during, and after fixation and after sectioning. Tension and length were recorded in the resting and contracted fiber before and during fixation. The I filaments could be traced to enter the A band between the A filaments on both sides of the I band, creating a zone of overlap which decreased linearly with stretch and increased with shortening. This is consistent with a sliding filament model. The decrease in the length of the A and I filaments during isometric contraction and the finding that fibers stretched to a sarcomere length of 3.7 µ still developed 30 per cent of the maximum tetanic tension could not be explained in terms of the sliding filament model. Shortening of the sarcomeres near the myotendinous junctions which still have overlap could account for only one-sixth of this tension, indicating that even those sarcomeres stretched to such a degree that there is a gap between A and I filaments are activated during isometric contraction (increase in stiffness). Shortening, too, was associated with changes in filament length. The diameter of A filaments remained unaltered with stretch and with isometric contraction. Shortening of 50 per cent was associated with a 13 per cent increase in A filament diameter. The area occupied by the fibrils and by the interfibrillar space increased with shortening, indicating a 20 per cent reduction in the volume of the fibrils when shortening amounted to 40 per cent.     

The structure of thick filaments on longitudinal sections of rabbit psoas muscle

By means of electron microscopy the longitudinal sections of chemically skinned fibres of rigorised rabbit psoas muscle have been examined at pH of rigorising solutions equal to 6, 7, 8 (I = 0.125) and ionic strengths equal to 0.04, 0.125, 0.34 (pH 7.0). It has been revealed that at pH 6.0 the bands of minor proteins localization in A-disks were seen very distinctly, while at pH 7.0 and I = 0.125 these bands can be revealed only by means of antibody labelling technique. At the ionic strength of 0.34 (pH 7.0) the periodicity of 14.3 nm in thick filaments was clearly observed, which was determined by packing of the myosin rods into the filament shaft and of the myosin heads (cross-bridges) on the filament surface. The number of cross-bridge rows in the filament equals 102. A new scheme of myosin cross-bridge distribution in thick filaments of rabbit psoas muscle has been suggested according to which two rows of cross-bridges at each end of a thick filament are absent. The filament length equals 1.64 +/- 0.01 micron. It has been shown that the length of thick filament as well as the structural organization of their end regions in rabbit psoas muscle and frog sartorius one are different.     

Sarcomere shortening in pressure overload hypertrophy

Sarcomere shortening during contraction was measured by using laser diffraction, in thin, rabbit right ventricular (RV) trabeculae from normal hearts (N) (n = 5) and from hearts subjected to RV pressure overload by pulmonary banding (H) (n = 5). Banding resulted in substantial RV hypertrophy after 2 wk. Hypertrophied preparations had the same resting muscle length (H = 3.15 +/- 0.29 mm) and resting sarcomere lengths (H = 2.16 +/- 0.005 micron) as the normal preparations (3.10 +/- 0.37 mm, 2.16 +/- 0.008 micron, respectively). Total tension at the peak of isometric twitches was the same as normal in the hypertrophied muscles (N = 8.06 +/- 1.20, H = 8.51 +/- 1.95 g/mm2). However, the amount of auxotonic sarcomere shortening was much less than normal in the hypertrophied preparations (N = 0.39 +/- 0.028, H = 0.19 +/- 0.034 micron; P less than 0.001). In isotonic contractions in which the ratio of muscle shortening to resting muscle length was the same in both the normal and hypertrophied muscles (ratio of 0.05 in both groups), the extent of sarcomere shortening relative to resting sarcomere length was less in the hypertrophied muscles than in the normal preparations (N = 0.14 +/- 0.01), H = 0.07 +/- 0.01; P less than 0.01). Series elasticity was the same as normal in the hypertrophied muscle P less than 0.05). Less auxotonic sarcomere shortening for a given level of isometric tension development and less isotonic sarcomere shortening per unit muscle shortening indicate that there is less than normal work per sarcomere during contraction in hypertrophied myocardium. These findings may have important implications for intracellular compensatory adaptation in pressure overload cardiac hypertrophy.