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(Circulation Research. 1995;77:199-205.)
© 1995 American Heart Association, Inc.

Articles

Osmotic Compression of Single Cardiac Myocytes Eliminates the Reduction in Ca²⁺ Sensitivity of Tension at Short Sarcomere Length

Kerry S. McDonald, Richard L. Moss

From the Department of Physiology, University of Wisconsin Medical School, Madison.

Correspondence to Kerry S. McDonald, PhD, Department of Physiology, University of Wisconsin, School of Medicine, 1300 University Ave, Madison, WI 53706.

	Abstract

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Abstract According to the Frank-Starling relation, cardiac output varies as a function of end-diastolic volume of the ventricle. The cellular basis of the relation is thought to involve length-dependent variations in Ca²⁺ sensitivity of tension; ie, as sarcomere length is increased in cardiac muscle, Ca²⁺ sensitivity of tension also increases. One possible explanation for this effect is that the decrease in myocyte diameter as muscle length is increased reduces the lateral spacing between thick and thin filaments, thereby increasing the likelihood of cross-bridge interaction with actin. To examine this idea, we measured the effects of osmotic compression of single skinned cardiac myocytes on Ca²⁺ sensitivity of tension. Single myocytes from rat enzymatically digested ventricles were attached to a force transducer and piezoelectric translator, and tension-pCa relations were subsequently characterized at short sarcomere length (SL), at the same short SL in the presence of 2.5% dextran, and at long SL. The pCa (-log[Ca²⁺]) for half-maximal tension (ie, pCa₅₀) increased from 5.54±0.09 to 5.65±0.10 (n=7, mean±SD, P<.001) as SL was increased from 1.85 to 2.25 µm. Osmotic compression of myocytes at short length also increased Ca²⁺ sensitivity of tension, shifting tension-pCa relations to [Ca²⁺] levels similar to those observed at long length (pCa₅₀, 5.68±0.11). These results support the idea that the length dependence of Ca²⁺ sensitivity of tension in cardiac muscle arises in large part from the changes in interfilament lattice spacing that accompany changes in SL.

Key Words: Ca²⁺ sensitivity • cardiac myocytes • isometric contraction

	Introduction

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An important mechanism in beat-to-beat optimization of myocardial performance is the enhancement of contractility in response to an increase in end-diastolic volume, ie, the Frank-Starling relation. The cellular mechanism by which volume and, consequently, muscle length regulate myocardial contraction is uncertain, although there is evidence suggesting that several factors may be involved (reviewed by Allen and Kentish¹ and Lakatta² ). For instance, the level of cardiac muscle activation by intracellular Ca²⁺ may be altered as a function of sarcomere length (SL). Consistent with this idea, a sustained decrease in the length of isolated papillary muscle produced a slow (10- to 20-minute) decline in the Ca²⁺ transient, which was temporally related to reductions in twitch tension.^{3 4} However, Ca²⁺ availability alone does not likely limit twitch tension, since the amplitude of the Ca²⁺ transient immediately after a decrease in muscle length was unchanged while tension was significantly reduced.^{3 4} Conceivably, the amount of Ca²⁺ that binds to troponin C and regulates cross-bridge interactions with actin may change as a function of muscle length. Consistent with this idea, some experimental evidence suggests that the affinity of troponin for Ca²⁺ is altered by changes in cardiac muscle length. For example, a sudden reduction in the length of skinned ferret papillary muscle bathed in aequorin resulted in a rapid and sustained increase in myoplasmic Ca²⁺.⁵ Also, the binding of isotopic Ca²⁺ to troponin in ventricular strips was greater at an SL of 2.3 µm than at 1.7 µm in the [Ca²⁺] range of 1.6 to 30 µm.^{6 7 8} In both these experiments, Ca²⁺ binding to troponin correlated more closely with muscle tension generation than muscle length per se, suggesting the possibility of mechanical feedback between force-generating cross-bridges and Ca²⁺ binding to troponin.

The cellular mechanism underlying the Frank-Starling relation also likely involves physical factors, as manifested by the well-characterized SL–maximum tension relation. In tetanically stimulated single skeletal muscle fibers, tension decreased when SL was reduced below optimum, ie, onto the ascending limb of the length-tension relation.⁹ The decrease in tension appears to result from double overlap of thin filaments such that thin filaments from the opposite side of the sarcomere interfere with cross-bridge binding or cross-bridges actually bind thin filaments of the opposite polarity. When SL was reduced below 1.6 µm, the rate of fall in tension was greater, which was likely due to a restoring force arising from thick filaments abutting the Z line. Similar length-tension relations have been obtained in maximally activated preparations of both skinned skeletal¹⁰ and cardiac¹¹ muscles, although the falloff of tension at very short lengths was less severe in cardiac muscle. Thus, length-dependent changes in tension-generating capability as a result of changes in filament geometry can account for, at most, only 20% of the nearly 50% variation in twitch contraction^{12 13} over an SL range of 1.8 to 2.3 µm, a range that encompasses most of the working range of living cardiac muscle.

Length-tension relations of striated muscle are altered considerably at [Ca²⁺] levels that yield submaximal tensions at optimum length.^{14 15 16 17 18 19} At low [Ca²⁺] levels, the SL for optimum tension development increases to >2.3 µm.^{14 16 17} Thus, as SL is increased, tension increases, indicating that Ca²⁺ sensitivity of tension is greater at long SLs. Consistent with this idea, the midpoint (ie, pCa₅₀) of relative tension-pCa relations shifted to higher pCa, ie, to lower [Ca²⁺], as SL was increased in skeletal and cardiac muscles.^{18 19 20 21 22}

Since the increase in myoplasmic Ca²⁺ during a twitch is transient and therefore submaximal for much of the time course of a twitch, the apparent decrease in Ca²⁺ sensitivity of tension at short lengths probably contributes significantly to the Frank-Starling relation. One possible explanation for the decrease in Ca²⁺ sensitivity of tension at short lengths is the increase in lateral separation of thick and thin filaments as SL is reduced,²³ which would decrease the likelihood of cross-bridge interaction in the zone of overlap between thick and thin filaments. In support of this idea, reducing the width of either skinned skeletal muscle fibers^{21 24} or rat trabeculae²⁵ by osmotic compression increases the Ca²⁺ sensitivity of tension at optimum SL. Thus, the purpose of the present study was to determine if changes in Ca²⁺ sensitivity of tension as a function of SL result from alterations in the diameter of myocytes and, consequently, interfilament lattice spacing. This hypothesis was tested by characterizing the length dependence of Ca²⁺ sensitivity of tension in single cardiac myocytes and determining whether osmotic compression of myocytes would eliminate the reduced Ca²⁺ sensitivity of tension observed at short SLs.

	Materials and Methods

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Myocyte Preparation
Single ventricular myocytes were obtained by enzymatic digestion of rat hearts, as previously described by Strang et al.²⁶ Myocytes were skinned by suspension for 6 minutes in a relaxing solution (mmol/L: free Mg²⁺ 1, KCl 100, EGTA 2, ATP 4, and imidazole 10, pH 7.0) containing 0.3% Triton X-100 (Pierce Chemical Co). The cells were then washed twice with fresh relaxing solution and stored on ice (4°C) for the day of the experiment.

The experimental apparatus and cell attachment procedure were similar to those described previously,^{26 27} with some modifications. The experimental apparatus consisted of a flat aluminum plate with three thermoelectric devices (Cambion, Midland-Ross Co) mounted along one edge for temperature control. The thermoelectric devices were cooled by a heat sink cooled with circulating water. The center portion of the plate was made of a separate stainless steel piece containing four circular chambers (diameter, 5 mm) with glass floors for the objective. Rapid solution changes were made by translating the plate laterally via a stage manipulator such that another solution-containing chamber was brought beneath the cell.

Single myocytes were attached with silicone adhesive (Dow Corning aquarium sealant) to glass micropipettes, which in turn were fastened to the active elements of a force transducer and a piezoelectric translator. The force transducer (model 403; compliance, 19 µm/mg, Cambridge Technology, Inc) and piezoelectric translator (Physik Instrument Co) were mounted to micromanipulators (No. MM-31, Narishige Scientific Instrument Laboratory) for precise three-way positioning. For measurements of total tension, the piezoelectric translator was used to impose length changes of up to 30 µm in <1 millisecond in order to achieve a zero-force baseline. The displacement of the translator was driven by a bipolar operational power supply/amplifier (Kepco Inc) controlled by a pulse-interval generator (series 1800, World Precision Instruments). The signals from the pulse generator and force transducer were recorded on a digital oscilloscope (model NIC-310, Nicolet Instrument Corp) and stored on magnetic disk for subsequent analysis.

Myocytes were observed through an inverted microscope (Carl Zeiss, Inc) having a 40x objective, 15x eyepiece, and a 1.6x intermediate lens. By use of a video camera (model WV-BL600, Panasonic) and a VHS recorder (model HR-S6600U, JVC), cell width, SL, and SL uniformity were monitored and recorded while the myocyte was relaxed and during each activation at 1680x magnification. SL along the length of the myocyte was measured by ruler from the image on the video monitor, which was calibrated at 1.69 mm/µm. At short SLs, the striation pattern during maximal activation was less clear than at long lengths (see Fig 1 for an example). The pattern during maximal activation was, on average, even less distinct at short length in the presence of 2.5% dextran. In fact, in three preparations, the striation pattern at short length could not be resolved during maximal activation in the presence of dextran, and thus SLs at 50% activation are reported in these cases. The exact reason for diminished clarity of the striation pattern at short length is unknown, but since changes in SL between relaxed and maximally activated conditions were very similar (reductions of <7%) whether the myocytes were at long or short length, it is unlikely that differential sarcomere shortening due to compliant ends is the cause. In the four myocytes for which SL could be visualized during maximal activation, the average ratio of SLs at 100% versus 50% activation was 0.99±0.04, validating the use of SL at 50% activation. For each myocyte, width was measured while the cell was relaxed, maximally activated, and during activations at 50% peak tension (P_o). In each condition, width was measured at three different fixed points along the length of the myocyte. The plane of focus for all width measurements was adjusted to obtain the sharpest contrast between the edge of the myocyte and the surrounding solution. The depth of field of the measurement plane was <12 µm, so it was not possible to routinely measure the depth of the myocytes.

View larger version (103K): [in this window] [in a new window]   Figure 1. Light photomicrographs of cardiac myocyte while relaxed (top) and during maximal activation at long (middle) and short (bottom) sarcomere lengths. For this preparation, relaxed sarcomere length was 2.35 µm, and sarcomere lengths during maximal activation were 2.21 and 1.88 µm at long and short lengths, respectively. The apparent change in distance between the two micropipettes in the top and middle panels is due to an optical artifact. The micropipettes are differentially out of focus in the two different photomicrographs. Bar=50 µm.

Skeletal Muscle Preparation
Two- to 3-month-old female Sprague-Dawley rats (250 to 350 g) were anesthetized with sodium pentobarbital (30 mg/kg body wt IP). Soleus muscles were removed and placed in ice-cold relaxing solution, and 8 to 10 bundles 1 to 2 mm in width (50 to 100 fibers) were dissected from regions throughout the muscle. Each bundle was tied with 4-0 suture to a glass capillary tube at approximately in situ length and placed in relaxing solution containing 50% glycerol (vol/vol) for storage at -20°C for up to 4 weeks. On the day of an experiment, a muscle bundle was placed in cold relaxing solution containing 0.5% Brij 58 (polyoxyethylene 20 cetyl ether, Sigma Chemical Co). The bundle was then transferred to a dissecting chamber containing cold relaxing solution, and a single fiber was isolated and mounted in an experimental apparatus similar to one previously described.¹⁰ Briefly, the ends of the fiber were placed in stainless steel troughs (25 gauge), which in turn were glued to styluses extending from the active elements of a force transducer and a DC torque motor (models 403 and 300Hz, respectively; Cambridge Technology, Inc). The ends were secured by overlaying a 0.5-mm length of 4-0 monofilament nylon suture, which was tied into the troughs with two loops of 10-0 monofilament suture. Muscle fiber force and length signals were digitized at 1000 Hz with a 12-bit A/D converter (AT-MIO-16F-5, National Instruments Corp), and each was displayed and stored on a personal computer by using custom software (LABVIEW for Windows, National Instruments Corp). Muscle-length changes were driven by computer-generated voltage commands to the torque motor via a 12-bit D/A converter (AT- MIO-16F-5, National Instruments Corp) by use of the custom software. Fiber width and SL were monitored and recorded at 1000x with a video camera and recorder mounted on an inverted microscope (model IMT-2, Olympus Instrument Co).

Solutions
Compositions of relaxing and activating solutions used in mechanical measurements were as follows (mmol/L): EGTA 7, free Mg²⁺ 1, imidazole 20, free ATP 4, and creatine phosphate 14.5, pH 7.0, along with various levels of [Ca²⁺] between 10^-9 mol/L (relaxing solution) and 10^-4.5 mol/L (activating solution) and sufficient KCl to adjust ionic strength to 180 mmol/L. Myocytes were osmotically compressed by adding dextran (molecular weight, 503 000; Sigma) to relaxing and activating solutions. Concentrated stocks of pCa 9.0 and 4.5 were prepared, and 2.5 g dextran/100 mL was added before dilution to final volume.²⁴

Experimental Protocol
Three tension-pCa relations were characterized for each cardiac myocyte. A tension-pCa relation was first characterized at short SL (1.85 µm). The myocyte was then bathed in pCa 9.0 solution containing 2.5% dextran, and a second tension-pCa relation was characterized. The myocyte was then washed several times with pCa 9.0 solution without dextran and stretched to long SL (2.25 µm), and a final tension-pCa relation was obtained. Control measurements at short lengths indicated that washing the myocytes reversed dextran-induced changes in Ca²⁺ sensitivity of tension. This sequence for obtaining tension-pCa relations was optimal for resolving the striation pattern at each stage of the protocol. Preliminary experiments revealed that several activations of the myocyte at long lengths made it difficult to return the myocyte to short length, and once returned to short lengths, the striation pattern during activations was less distinct than when measurements were made before extension. For soleus fibers, two tension-pCa relations were characterized: the first one was at short SL; the second, at long SL. Tension-pCa relations were characterized by first maximally activating the cell and subsequently transferring the cell into a series of submaximal pCa solutions. At each pCa, a steady tension was allowed to develop, and the cell was rapidly slackened to determine total tension (Fig 2). The amount of active tension generated at each pCa was calculated as the difference between total tension and relaxed tension, which was assessed by slackening the cell while it was in the relaxed state. To determine any decline in tension-generating capability, the cell was maximally activated at the beginning, sometimes in the middle, and at the end of the protocol to obtain each tension-pCa relation. For a given curve, all cells maintained at least 80% of initial maximal tension. Tensions in submaximally activating solutions were expressed as fractions of P_o measured at the same SL. The P_o value used to normalize submaximal tensions was obtained by linear interpolation between successive maximal activations.

View larger version (28K): [in this window] [in a new window]   Figure 2. Representative force traces (A) and graph of tension-pCa relations (B) for a myocyte at long (2.26 µm) and short (1.90 µm) sarcomere lengths. The pCa50 values, ie, midpoint for half-maximal activation, were 5.69 and 5.55 at long and short lengths, respectively. P indicates tension; Po, peak tension.

Tension-pCa data were fit by computer using least-squares regression analysis with the following equation:

where P_r is tension as a fraction of P_o and n is the Hill coefficient. The form and midpoint (pCa₅₀) of the tension-pCa relation were determined by Hill plot analysis of the data.²⁸ Two separate straight lines were fitted to tension data above and below 0.5 P_o by least-squares analysis using the following equation:

where k is pCa₅₀. The slopes of the two phases of the Hill plot, above and below pCa₅₀, were n₁ and n₂, respectively.

Statistical Analysis
A one-way repeated-measures ANOVA was used to compare myocyte dimensions and tension-pCa relations between long, short, and short SL plus 2.5% dextran groups. Paired t tests were used as post hoc tests to estimate differences among means. To determine whether there were significant differences in soleus muscle fiber dimensions and tension-pCa relations as a function of SL, paired t tests were used. All values are mean±SD, and a value of P<.05 was chosen as indicating significance.

	Results

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Length Dependence of Ca²⁺ Sensitivity of Tension in Single Myocytes and the Effect of Osmotic Compression
Force traces and the corresponding tension-pCa relations are shown in Fig 2 for a single myocyte at long and short SLs. The peak total tensions generated by this myocyte were 10.8 nN at short SL and 15.5 nN at long SL, and passive tensions were 0.85 and 2.4 nN, respectively. In this case, an increase of 0.36 µm in SL resulted in a significant increase in Ca²⁺ sensitivity of tension, since the Ca²⁺ required for half-maximal activation (pCa₅₀) increased 0.14 pCa units (5.55 to 5.69).

Fig 3 shows cumulative (n=7) tension-pCa relations at long SL, short SL, and short SL in the presence of 2.5% dextran. Mean pCa₅₀ was 5.54±0.09 at short SL, and an increase in SL significantly shifted pCa₅₀ to lower [Ca²⁺] levels (pCa₅₀, 5.65±0.10 at long SL; P<.001). Treatment with 2.5% dextran at short length also shifted the tension-pCa relation to a lower [Ca²⁺] (ie, greater sensitivity to Ca²⁺) and very near to the mean relation obtained at long SL. Mean pCa₅₀ was 5.68±0.11 after treatment with 2.5% dextran (P<.001 versus short SL).

View larger version (14K): [in this window] [in a new window]   Figure 3. Graphs showing mean tension-pCa relations (left) and Hill plots (right) for cardiac myocytes at long length, short length, and the same short length plus 2.5% dextran. Values are mean±SD. Pr indicates relative tension (P/Po, where P is tension and Po is peak tension). Cell characteristics and Ca2+ sensitivity of tension values are presented in Table 1.

A summary of myocyte length, width, SL, force, Hill coefficients, and pCa₅₀ for each of the three conditions is presented in Table 1. Previous studies of single skinned skeletal muscle fibers have demonstrated that fiber width is reduced by increasing fiber length and by treatment with dextran-containing solutions.^{21 29} Our results indicate that the width of single skinned cardiac myocytes responds to length changes and exposure to dextran-containing solutions in a manner similar to that demonstrated by skeletal muscle fibers. Increasing SL from 1.85 to 2.25 µm as well as treatment with 2.5% dextran significantly reduced myocyte width. Mean myocyte width decreased by 10.3% when SL was increased by 0.4 µm and by 6.5% after dextran treatment at short lengths.

View this table: [in this window] [in a new window]   Table 1. Myocyte Dimensional Characteristics, Hill Coefficients, and pCa50 Values

Several studies have observed enhancement of maximum tension after treatment of skinned skeletal fibers and cardiac trabeculae with moderate concentrations (3% to 5%) of dextran.^{21 25 29} We observed that osmotic compression of single skinned myocytes with 2.5% dextran caused a significant increase in peak tension at short SL. The first peak tension value obtained after dextran treatment (8.4±2.2 nN) was always greater than the last maximum tension value obtained at short length (7.4±2.2 nN) just before dextran exposure. Peak tension at long SL (10.6±3.1 nN) was significantly greater than that obtained at short length both in the presence and absence of dextran. The paired ratio of maximum tension at short SL+2.5% dextran versus short SL (last value before dextran exposure) was 1.15±0.05. The paired ratio of maximum tension at long versus short SLs was 1.42±0.14 when using the last short value before dextran exposure and 1.27±0.12 when taking the first maximum activation at short length.

The slopes of the lines fitted to the data following Hill plot transformation provide an index of the apparent cooperativity of tension generation. Neither slope from the two phases of the Hill plot was significantly affected by SL or osmotic compression, at least within the variability of the data.

Length Dependence of Ca²⁺ Sensitivity of Tension in Slow Skeletal Muscle Fiber
Reductions in muscle length are associated with reduced binding of isotopic Ca²⁺ to troponin in cardiac muscle^{6 7 8} but not slow soleus muscle,⁸ even though the isoform of troponin C is the same in both muscles.³⁰ To examine the role of length-dependent Ca²⁺ binding in conferring length dependence of Ca²⁺ sensitivity of tension, we compared the Ca²⁺ sensitivity of tension as a function of length in soleus fibers with that in cardiac myocytes. Summaries of soleus fiber length, width, SL, force, Hill coefficients, and pCa₅₀ values are presented in Table 2. Peak force at long and short SL was 240±77 and 209±70 nN (ratio of long to short, 1.16±0.13), respectively. Mean pCa₅₀ at short SL (1.88±0.02 µm) was 5.76±0.03, and an increase in SL to 2.31±0.06 µm shifted pCa₅₀ to 5.86±0.06 (P<.001), a shift very similar to that observed in cardiac myocytes (compare Fig 3 and Fig 4). The long SL versus short SL pCa₅₀ ratios were 1.018±0.008 in soleus fibers and 1.020±0.006 in cardiac myocytes.

View this table: [in this window] [in a new window]   Table 2. Soleus Fiber Dimensional Characteristics, Hill Coefficients, and pCa50 Values

View larger version (12K): [in this window] [in a new window]   Figure 4. Graphs showing mean tension-pCa relations (left) and Hill plots (right) for soleus fibers at long and short sarcomere lengths. Values are mean±SD. Pr indicates relative tension (P/Po, where P is tension and Po is peak tension). Cell characteristics and Ca2+ sensitivity of tension values are presented in Table 2.

	Discussion

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According to the Frank-Starling relation, cardiac output depends on the end-diastolic volume of the ventricle, which is manifest in isolated myocardium in the rapid falloff of twitch tension as muscle length is reduced.^{12 13 31 32} Three mechanisms likely account for the majority of the steepness of the length-twitch tension relation. First, since isolated cardiac muscle does not ordinarily assume an SL of <1.9 µm at rest, measurements of twitch tension at shorter SLs require that the resting muscle be slackened so that these lengths can be attained during the subsequent twitch. In these cases, a large fraction of the myoplasmic Ca²⁺ transient is taken up by shortening of the muscle to the desired length, so that relatively little of the Ca²⁺ transient would remain for isometric tension development. Second, shortening induces dissociation of Ca²⁺ from troponin as the slackened muscle shortens during a twitch. In mammalian myocardium, a sudden reduction in muscle length during a twitch contraction results in the appearance of extra Ca²⁺ in the myoplasm.^{3 5 33} Thus, the level of activation of the thin filaments by the time of peak tension (ie, after shortening) is likely to be lower than would be predicted on the basis of the Ca²⁺ transient alone. Third, since the increase in myoplasmic Ca²⁺ during a twitch is transient and therefore submaximal for much of the twitch, the reduced Ca²⁺ sensitivity of tension at short lengths probably contributes significantly to the falloff of twitch tension.

We have characterized the effect of SL on Ca²⁺ sensitivity of tension in single cardiac myocytes. In the present study, consistent with earlier work using whole cardiac muscle preparations,^{18 19 20} we found that Ca²⁺ sensitivity of tension was greater at long SLs. The -log[Ca²⁺] required for 50% activation (ie, pCa₅₀) increased from 5.54±0.09 to 5.65±0.10 as SL was increased from 1.85 to 2.25 µm. Previous experiments using whole heart muscle preparations observed shifts in pCa₅₀ of 0.12 to 0.31 pCa units with similar changes in SL. The greater length dependence of Ca²⁺ sensitivity of tension in whole muscle preparations may be due to additional restoring forces arising from intercellular connections and extracellular connective tissue that are absent in single myocytes (reviewed by Lakatta² ).

The focus of the present study was to determine mechanisms underlying length dependence of Ca²⁺ sensitivity of tension in cardiac muscle. We investigated the hypothesis that increases in myocyte diameter and concomitant increases in interfilament lattice spacing cause decreased Ca²⁺ sensitivity of tension at short SL. Our finding that osmotic compression of myocytes at short SL reduced myocyte width and significantly increased the Ca²⁺ sensitivity of tension indicates that, in fact, the length dependence of Ca²⁺ sensitivity of tension in cardiac muscle arises in large part from changes in interfilament lattice spacing that accompany changes in SL.

We did observe a slight mismatch between the shift in Ca²⁺ sensitivity and the change in myocyte width after osmotic compression versus myocyte lengthening. Osmotic compression of myocytes at short length resulted in shrinkage to 93% of the width before compression, whereas increases in SL to 2.25 µm decreased width to 90% of the precompressed value, yet the shift in Ca²⁺ sensitivity of tension was similar under both conditions. The exact reason(s) for this mismatch is unknown. One possible explanation is that the shape of the myocyte was altered differently by mechanical stretch compared with osmotic compression with dextran. A differential shape change would result in varying the ratios of width to cross-sectional area at long lengths versus short lengths and would thus alter estimations of filament lattice spacing indexed by myocyte width measurements. Regardless of the exact reason for the small dextran-induced overshoot of Ca²⁺ sensitivity of tension with respect to myocyte diameter, it is evident that reduced Ca²⁺ sensitivity of tension at short length is mostly due to increases in myofilament lattice spacing.

The molecular mechanism by which changes in myocyte length and, thus, interfilament lattice spacing alters Ca²⁺ sensitivity of tension has yet to be determined. A simple explanation is that at a given [Ca²⁺], more Ca²⁺ is bound to the thin filaments at long lengths, thereby increasing the number of tension-generating cross-bridges. Greater Ca²⁺ binding to troponin C at long versus short SLs may occur as a result of changes in length and/or lattice spacing per se, changes in force as a function of length and/or lattice spacing, or increased myoplasmic charge density that accompanies lattice compression, thereby altering the concentration of cations surrounding the thin filament. Hofmann and Fuchs^{6 7} have obtained direct evidence that the binding of Ca²⁺ to troponin C is greater at long (2.3 µm) versus short (1.6 µm) SLs. Also, Allen and Kentish⁵ showed that a rapid decrease in muscle length resulted in an increase in myoplasmic [Ca²⁺]. In both cases, the amount of Ca²⁺ bound appeared to be related to force and thus the number of cross-bridges bound to actin rather than to length per se. Since strongly bound cross-bridges are known to enhance Ca²⁺ binding to troponin C,^{34 35} it is difficult to know whether length-dependent changes in Ca²⁺ binding are a cause or a result of length-dependent changes in tension. Recently, Wang and Fuchs⁸ reported that reductions in length and force similar to those that reduced Ca²⁺ binding in cardiac muscle had no effect on binding of Ca²⁺ in slow skeletal muscle. Our results indicate that changes in length alter Ca²⁺ sensitivity of tension similarly in both cardiac and slow skeletal muscle. This may mean that the greater Ca²⁺ binding at long lengths in cardiac muscle has little significance in conferring greater Ca²⁺ sensitivity of tension and is more a consequence of the change in force associated with length changes. Whether this is the case or whether the length dependence of Ca²⁺ binding plays a more important role in length-dependent activation in cardiac muscle requires further studies.

A potentially important mechanism of length dependence of Ca²⁺ sensitivity of tension that could be common to all striated muscles is that as the lateral separation between thick and thin filaments decreases, the likelihood of cross-bridge interaction in the zone of overlap between filaments increases. A greater probability of binding would increase tension directly by increasing the number of cross-bridges bound and indirectly by recruiting even more cross-bridges via cross-bridge–induced cooperative activation of the thin filament.³⁶ The resultant increase in the number of cross-bridges bound per unit length of thick/thin filament overlap would explain the increase in Ca²⁺ sensitivity of relative tension at longer SLs, even at SLs at which maximum absolute tension is observed to decrease.^{14 15 16 22} However, in at least one model, an increase in the probability of cross-bridge binding induced by increasing the ratio of apparent rate constants for attachment and detachment resulted in a greater Hill coefficient for the tension-pCa relation,³⁷ a result that is inconsistent with our finding that Hill coefficients were not significantly altered by osmotic compression.

	Acknowledgments

 
This study was supported by a grant from the American Heart Association (Dr Moss) and National Institutes of Health postdoctoral fellowship HL-08755 (Dr McDonald). The authors thank Scott Stoker for technical assistance with the experimental preparation.

Received October 17, 1994; accepted March 27, 1995.

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