This Article Abstract Full Text (PDF) All Versions of this Article: 90/1/59    most recent hh0102.102269v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Konhilas, J. P. Articles by de Tombe, P. P. Search for Related Content PubMed PubMed Citation Articles by Konhilas, J. P. Articles by de Tombe, P. P. Related Collections Structure Contractile function Calcium cycling/excitation-contraction coupling Cell biology/structural biology

(Circulation Research. 2002;90:59.)
© 2002 American Heart Association, Inc.

Cellular Biology

Myofilament Calcium Sensitivity in Skinned Rat Cardiac Trabeculae

Role of Interfilament Spacing

John P. Konhilas, Thomas C. Irving, Pieter P. de Tombe

From the Department of Physiology and Biophysics and Cardiovascular Sciences Program (J.P.K., P.P.d.T.), College of Medicine, University of Illinois at Chicago, Ill; and CSRRI and the Department of Biological, Chemical and Physical Sciences (T.C.I.), Illinois Institute of Technology, Chicago, Ill.

Correspondence to Pieter P. de Tombe, PhD, Department of Physiology and Biophysics (M/C 902), College of Medicine, University of Illinois at Chicago, 900 S Ashland Ave, Chicago, IL 60607-7171. E-mail pdetombe{at}uic.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The increase in myofilament Ca²⁺ responsiveness on an increase in sarcomere length (SL) is, in part, the cellular basis for Frank-Starling’s law of the heart. It has been suggested that a decrease in myofilament lattice spacing (LS) in response to an increase in SL underlies this phenomenon. This hypothesis is supported by previous studies in which reduced muscle width induced by osmotic compression was associated with an increase in Ca²⁺ sensitivity, mimicking those changes observed with an increase in SL. To evaluate this hypothesis, we directly measured LS by synchrotron x-ray diffraction as function of SL in skinned rat cardiac trabeculae bathed in 0% to 6% dextran solutions (MW 413 000). We found that EC₅₀, [Ca²⁺] at which force is half-maximal, at SL between 1.95 and 2.25 µm did not vary in proportion to LS when 3% or 6% dextran solutions were applied. We also found that moderate compression (1% dextran) of skinned trabeculae at SL=2.02 µm reduced LS (LS=42.29±0.14 nm) to match that of uncompressed fibers at a long SL (SL=2.19 µm; LS=42.28±0.15 nm). Whereas increasing SL from 2.02 to 2.19 µm significantly increased Ca²⁺ sensitivity as indexed by the EC₅₀ parameter (2.87±0.11 µmol/L to 2.52±0.12 µmol/L), similar reduction in myofilament lattice spacing achieved by compression with 1% dextran did not alter Ca²⁺ sensitivity (2.87±0.10 µmol/L) at the short SL. We conclude that alterations in myofilament lattice spacing may not be the mechanism that underlies the sarcomere length–induced alteration of calcium sensitivity in skinned myocardium.

Key Words: sarcomere length • x-ray diffraction • osmotic compression • regulation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The heart regulates ventricular output in response to changes in ventricular filling, a mechanism known as Frank-Starling’s law of the heart. This mechanism has two components¹: a fast component caused by changes in myofilament calcium sensitivity,^2,3 and a slow component caused by a change in the calcium transient.⁴ In this study, we are concerned only with the fast component that is responsible for the beat-to-beat changes in ventricular output in response to ventricular filling.

It is not clear how the information concerning sarcomere length (SL) is transmitted to the myofilaments. Although thin filament proteins are not ruled out as potential regulators of length-dependent activation,⁵ a more unifying hypothesis suggests that the lateral spacing between actin and myosin influences crossbridge reactivity independent of the primary events involving Ca²⁺ binding and subsequent thin filament activation.^6–8 As the interfilament spacing is reduced with increased SL,^9–11 it is hypothesized that the probability of strong crossbridge formation is enhanced. Facilitated by the highly cooperative nature of myofilament activation, the sensitivity of the myofilaments to Ca²⁺ would thus be increased at longer SL. In support of this hypothesis, shrinkage of the myofilament lattice by osmotic compression using high molecular weight moieties has been shown to increase myofilament Ca²⁺ sensitivity at short SL, mimicking the impact of increased SL on myofilament lattice spacing and Ca²⁺ sensitivity.^{6–8,12–14} The extent of shrinkage of the myofilament lattice by osmotic compression in those studies, however, was estimated from the reduction in muscle width. Muscle width in skinned preparations may not be strictly proportional to myofilament lattice spacing,¹⁵ an observation that we confirm in the present study in skinned isolated rat myocardium.

Accordingly, we set out to obtain a direct comparison between myofilament Ca²⁺ sensitivity and myofilament lattice spacing in skinned rat cardiac trabeculae under conditions of varying degrees of osmotic compression induced by dextran. Measurements of Ca²⁺ sensitivity were obtained at 3 SLs, and these measurements were correlated with direct measurement of myofilament lattice spacing obtained via synchrotron x-ray diffraction. We found that Ca²⁺ sensitivity did not vary in proportion to myofilament lattice spacing in osmotically compressed trabeculae. Furthermore, when the myofilament lattice was osmotically compressed to match the reduced lattice spacing as induced by increased SL, myofilament Ca²⁺ sensitivity was not affected. These findings suggest, therefore, that alterations in myofilament lattice spacing may not be the primary mechanism that underlies the sarcomere length–induced alteration of calcium sensitivity in skinned myocardium. A preliminary report of these studies has been published previously.¹⁶

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Muscle Preparation and Experimental Protocol
Rats (LBNF-1 220 to 280 g) were anesthetized (sodium pentobartbital 50 mg/kg BW IP injection), and the hearts were rapidly excised and retrogradely perfused with a modified Krebs-Henseleit (K-H) solution with propranolol (5 µmol/L), to block nonspecific ß-adrenergic activation, and carbamyl choline (10 µmol/L), to enhance phosphatase activity.¹⁷ Trabeculae were dissected from the right ventricular free wall and demembranated overnight with 1% Triton X-100 in standard relaxing solution (EGTA=10 mmol/L; ionic strength=180 mmol/L). All solutions contained the following (in mmol/L): 10 phosphocreatine, 100 N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 0.1 leupeptin, 0.1 phenylmethylsulfonyl fluoride (PMSF), 1 dithiothreitol (DTT), and 4 U/mL creatine phosphokinase. The free Mg²⁺ and Mg-ATP concentration were calculated at 1 and 5 mmol/L, respectively. Relaxing and activating solutions were appropriately mixed to obtain a range of free [Ca²⁺] at 15°C. Trabeculae (n=22; 0% dextran) were 2 to 3 mm in length, 136±10 µm in width, and 104±10 µm in thickness. Ca²⁺-force relationships were determined at three sarcomere lengths (SL=1.95 µm, 2.10 µm, and 2.25 µm). Muscle length was adjusted to maintain constant SL throughout the contraction.¹⁸ SL was determined from the first order He-Ne laser light diffraction band. Steady-state force was recorded at each SL (Figure 1). Muscles that did not maintain 90% of maximal activation on completion of the experiment or that lost the laser diffraction band were discarded. In some experiments, 1%, 3%, or 6% (wt/volume) dextran (Sigma, 413 kDa) was added to the solutions (equilibration >30 minutes). X-ray diffraction experiments were performed as described previously.¹¹

View larger version (18K): [in this window] [in a new window]   Figure 1. Method used to measure active force development under maximal and submaximal activation in rat cardiac trabeculae. Data were recorded at 0% dextran (left), 3% dextran (middle), and 6% dextran (right). A, Original recordings of force, normalized as force/unit area, at varied free [Ca2+] (as indicated in µmol/L) for each dextran concentration (SL=2.25 µm). A quick release was used to determine zero force level. Calibrations as indicated. B, Ca2+-force relationship, constructed from data obtained as illustrated in A. The solid lines indicate the Hill fit to the data obtained at the indicated dextran concentration; the dotted lines represents the Hill fit obtained at 0% (middle) or 0% and 3% (right).

Data Analysis
Each individual Ca²⁺-force relationship was fit to a modified Hill equation: F_rel=[Ca²⁺]ⁿ/(EC₅₀ⁿ+[Ca²⁺]ⁿ); F_rel=relative force, EC₅₀=[Ca²⁺] at which force is half-maximal, n=Hill coefficient. Separation of the equatorial 1,0 or 1,1 x-ray diffraction reflections was measured from detector images using the program FIT2D¹⁹ and converted to d₁₀ lattice spacing using Bragg’s Law, which can then be converted to the interthick filament spacing by multiplying d₁₀ by 2/3. The differences among EC₅₀, EC₅₀, LS, and LS for each group at each SL were analyzed with a one-way ANOVA followed by a Student’s t test with a post hoc Bonferroni correction to assess differences among mean values. All data are shown as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myofilament Ca²⁺ Sensitivity
To assess the effect of sarcomere length on myofilament Ca²⁺ sensitivity, Ca²⁺-force relationships were determined at 3 SLs (1.95, 2.10, and 2.25 µm) in each of the groups studied (0%, 3%, and 6% dextran solution). Original recordings of force from a typical experiment are illustrated in Figure 1. Steady-state force was measured at each SL during each activating cycle; the zero force level was identified by instituting a quick ramp shortening in muscle length just before introduction of relaxing solution into the bath (active force was calculated as the difference between total force and relaxed force). Figure 1A shows a series of such quick length-release steps recorded at varying levels of activation (Ca²⁺ in µmol/L indicated on the right of each tracing) for trabeculae bathed with a 0%, 3%, or 6% dextran solution (SL=2.25 µm). A final maximal activation was administered to assess muscle preparation integrity. The relationship between active force development and Ca²⁺ concentration ([Ca²⁺]) was fit to the Hill equation (see Materials and Methods) at each dextran concentration as is illustrated in Figure 1B. Application of either 3% or 6% dextran induced a significant increase in myofilament Ca²⁺ sensitivity (Figure 1B and pooled data in Figure 2). The average parameters of the Hill fit obtained in each individual trabecula are summarized in the Table. Increasing SL from 1.95 to 2.10 µm and then to 2.25 µm caused a leftward shift of the Ca²⁺-force relationship (Figure 2) and a significant decrease in the EC₅₀ parameter for each experimental group (Table). Addition of 3% dextran (wt/volume) significantly increased Ca²⁺ sensitivity at all 3 SLs from the control (0% dextran) group (Figure 2A). Further addition of dextran from 3% to 6% slightly increased Ca²⁺ sensitivity at all SLs, albeit not significantly (Figure 2B; Table). Neither changes in SL nor changes in dextran concentration had an appreciable affect on the steepness of the Ca²⁺-force relationship (Hill coefficient; Table).

View larger version (18K): [in this window] [in a new window]   Figure 2. Ca2+-dependent force development in skinned cardiac trabeculae with and without dextran in the solutions. A, Ca2+-force relationship at varied SL (1.95 µm , 2.10 µm , and 2.25 µm ) in the absence (open symbols) or presence (filled symbols) of 3% dextran (n=7 to 8). B, Ca2+-force relationship at varied SL (1.95 µm , 2.10 µm , and 2.25 µm ) in the presence of 3% (open symbols) or 6% (filled symbols). Lines indicate the Hill fit to the data obtained at the indicated sarcomere length and dextran concentration.

View this table: [in this window] [in a new window]   Table 1. Average Hill Fit Parameters of the Ca2+-Force Relationships Under Conditions of Varied Dextran Concentration and Sarcomere Length (SL)

Although application of dextran caused a general increase in myofilament Ca²⁺ sensitivity at any SL, the length-dependent shift of the Ca²⁺-force relationship appeared not to be altered by dextran treatment. We quantified this by computing EC₅₀, the difference between EC₅₀ at the short and long sarcomere length in each muscle (SL=1.95 and 2.25 µm, respectively). Indeed, EC₅₀ (in µmol/L) was unaffected by dextran treatment (Table and Figure 4B). Previous studies have employed pCa₅₀ (-log[EC₅₀]) to index calcium sensitivity.^{6–8,12–14} Employing this method also revealed no statistically significant difference in length-dependent activation (Table). Ca²⁺-saturated maximum force decreased 20%, on average, with a reduction in SL from 2.25 to 1.95 µm in each experimental group, consistent with previous reports.³

View larger version (26K): [in this window] [in a new window]   Figure 4. A, Average myofilament lattice spacing as a function of sarcomere length in skinned cardiac trabeculae bathed in either a 0% (), 3% (), or 6% () dextran solution (n=8 to 10). The relationships were obtained by averaging the data into 0.05-µm wide sarcomere length bins. B, Average impact of SL on myofilament lattice spacing (LS) and Ca2+ sensitivity (EC50) at each dextran concentration. See text for details. Significant differences from control group (0% dextran), *P<0.01; P<0.01 from 3% dextran group.

Application of dextran may affect maximum force development.^8,20,21 Accordingly, we directly determined the effect of dextran application on maximum force in a separate group of trabeculae at SL=2.20 µm (n=6). We found that consecutive application of 1%, 3%, and 6% dextran increased maximal force development, on average, by 13.3±4.0%, 12.2±4.6%, and 11.9±5.2%, respectively, compared with that in trabeculae in the absence of dextran (P<0.01). This observation is consistent with previous reports.^7,8,21

Osmotic Compression and Myofilament Lattice Spacing
We simultaneously measured myofilament lattice spacing (LS) and SL in skinned cardiac trabeculae (n=8 to 10 for each group studied) using synchrotron x-ray diffraction.¹¹ Figure 3 illustrates typical x-ray diffraction patterns obtained in trabeculae at SL=2.1 µm in relaxing solutions containing 0%, 3%, or 6% dextran. There was an evident outward displacement of the reflections with increasing dextran concentration in the solution, indicative of a reduction in myofilament lattice spacing. Figure 4A shows that LS was an inverse function of SL at each dextran concentration (n=8 to 10). Furthermore, LS was reduced at every SL in trabeculae immersed in a 3% dextran solution compared with control trabeculae, and it was further reduced in trabeculae that were bathed in a 6% dextran solution. At SL=2.10 µm, LS was significantly reduced (P<0.001) with a 3% dextran solution (37.9±0.19 nm) from control (42.6±0.20 nm); adding 6% dextran to the relaxing solution further reduced LS (34.6±0.12 nm) significantly from both the control and the 3% dextran group at this SL.

View larger version (27K): [in this window] [in a new window]   Figure 3. Typical x-ray diffraction patterns obtained in skinned rat cardiac trabeculae in the absence or presence of 3% or 6% dextran in the bathing solution at SL=2.10 µm. A, CCD images of the diffraction patterns. X-ray diffraction by the myofilament lattice gives rise to 2 sets of symmetrical, equatorial reflections, delineated by the solid vertical lines. B, Intensity profiles of the 2-dimensional CCD images shown in A. Arrows indicate the peak intensities, corresponding to the 1,0 and 1,1 reflections. These clearly resolved reflections or intensity peaks allowed lattice spacing to be determined within 0.25% accuracy, equivalent to a myofilament lattice spacing resolution of 0.1 nm.

The impact of changes in SL on myofilaments lattice spacing, as reflected by the slope of the relationships displayed in Figure 4A, was markedly reduced at 3% and 6% dextran. That is, LS became a less sensitive function of SL with increasing amounts of dextran in the solution. To quantify this observation, we defined the decrease in LS when SL was increased from 1.95 to 2.25 µm as LS in each muscle. The average LS, as well as the EC₅₀ parameters are shown in Figure 4B. LS was greatest in the control trabeculae (1.67±0.16 nm), was markedly reduced in trabeculae bathed in the 3% dextran solution (0.70±0.04 nm), and reduced even further in trabeculae bathed in a 6% dextran solution (0.47±0.04 nm). Each of these values was significantly different from the other (P<0.01). Thus, length-dependent activation, as indexed by either the EC₅₀ or the pCa₅₀ parameter, was unaffected by dextran compression, whereas changes in myofilament lattice spacing (LS) induced by changes in SL were significantly reduced on compression by 3% or 6% dextran. LS remained inversely proportional to SL under all levels of dextran compression. Therefore, these results indicate that the impact of interfilament spacing on myofilament Ca²⁺ sensitivity is not constant but rather varies with the overall compression state of the cardiac sarcomere.

Ca²⁺ Sensitivity With Moderate Amounts of Osmotic Compression
At the outset of the present study, we choose dextran concentrations consistent with previous reports that used either 2.5%⁷ or between 0% to 5%^8,14,21 dextran concentrations. Indeed, the Ca²⁺ sensitivity of control trabeculae at the long SL was approximately matched by application of 3% dextran at the short SL (see Table). We found, however, that under our conditions, both 3% and 6% dextran reduced myofilament lattice spacing beyond the physiological effect of an increase in SL in control, uncompressed skinned trabeculae. That is, application of 3% dextran compressed the myofilament lattice beyond that observed at even the largest SL under control conditions (Figure 4). Therefore, to specifically test the notion that lattice spacing is the primary determinant for length-dependent myofilament Ca²⁺ sensitivity in skinned myocardium, we performed an additional set of experiments. The results are summarized in Figure 5. Skinned trabeculae were treated with 1% dextran to osmotically compress the myofilament lattice at a short SL so as to precisely match that of control, uncompressed trabeculae at a longer SL. Treatment of skinned cardiac trabeculae at a SL=2.02 µm with 1% dextran reduced LS to 42.29±0.14 nm, a value that was equivalent to LS at SL=2.19 µm in the untreated trabeculae (42.28±0.15 nm). Note that the LS at SL=2.02 µm with 0% dextran was 43.02±0.21 nm. Average Ca²⁺ sensitivities, as indexed by the EC₅₀ parameter, under these conditions are shown in the bar graph inset of Figure 5. Increasing SL from 2.02 µm to 2.19 µm resulted in a significant decrease in EC₅₀, consistent with the results shown in Figure 2 and Table. However, application of 1% dextran at SL=2.02 µm did not significantly affect Ca²⁺ sensitivity, despite the reduction in myofilament lattice spacing (whether indexed by EC₅₀ or pCa₅₀).

View larger version (20K): [in this window] [in a new window]   Figure 5. Myofilament Ca2+-force relationship at 2 sarcomere lengths (SL) and moderate dextran compression (n=7). Lines indicate the Hill fit to the data obtained at SL=2.19 µm () and 2.02 µm () without (open symbols) and with 1% dextran (closed symbols) in the solutions. Inset, Average myofilament Ca2+ sensitivity, as indexed by the EC50 parameters. EC50 in the absence of dextran was 2.87±0.11 and 2.52±0.12 µmol/L at SL=2.02 µm and SL=2.19 µm, respectively (pCa50 values were 5.54±0.01 and 5.60±0.01, respectively). Application of 1% dextran at SL=2.02 µm resulted in EC50=2.87±0.10 µmol/L (pCa50=5.54±0.01), values that are not significantly different from the control EC50 at SL=2.02 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The notion that the interfilament spacing constitutes the molecular length sensor is a prominent feature of current theories regarding the mechanism that underlies Frank-Starling’s law of the heart.^{6–8,12–14,22} The most compelling evidence in support of this theory comes from studies in which shrinkage of the myofilament lattice in skinned muscle preparations by osmotic compression is accompanied by increased Ca²⁺ responsiveness of the contractile apparatus.^6–8,12,13

The impact of sarcomere length (SL) on myofilament Ca²⁺ sensitivity, as indexed by either by the EC₅₀ or the pCa₅₀ parameter (Table), was unaffected by application of dextran, whereas the slope of the relationship between myofilament lattice spacing and sarcomere length (LS) was considerably reduced with increasing dextran concentration in the bathing solutions (Figure 4). This reduction in the length dependence of myofilament lattice spacing would be expected to lead to a reduction of the length dependence of myofilament activation. Osmotic compression in this study, however, did not affect the length dependence of the myofilament Ca²⁺ sensitivity in rat trabeculae, consistent with a recent report on rat ventricular myocytes.²³ Other studies have reported a reduced length dependency on compression of the myofilament lattice by dextran.^8,12 In those studies, Ca²⁺ sensitivity was indexed by pCa₅₀. It should be noted that the relation between pCa and [Ca²⁺] is nonlinear; therefore, the choice of parameter to index myofilament Ca²⁺ sensitivity could potentially affect data interpretation. Because reaction rates are usually proportional to the free concentration of a substrate, we believe that the use of the EC₅₀ parameter to index myofilament Ca²⁺ sensitivity is more appropriate than pCa₅₀. However, this factor alone cannot explain the different findings because neither the EC₅₀ nor the pCa₅₀ parameter was significantly affected by osmotic compression in the present study. The precise reason for the discrepancies between previous studies^8,12 and our current study is unclear; a species specific difference in the effect of dextran on length-dependent activation may underlie these results. Clearly, this issue requires further study.

Our data, using 3% and 6% dextran suggest that, at a minimum, the interfilament spacing theory must be amended to include a variable impact of myofilament lattice spacing on myofilament Ca²⁺ sensitivity, depending on the overall extent of compression of the myofilament lattice. The increase in Ca²⁺ sensitivity on the decrease in myofilament lattice spacing in the 3% dextran solution is consistent with the interfilament spacing hypothesis (Figures 2 and 4; Table). However, the further reduction in myofilament lattice spacing in 6% dextran without a further increase in Ca²⁺ sensitivity cannot readily be explained by this theory. The effect of dextran treatment on myofilament Ca²⁺ sensitivity has previously been shown to be limited to approximately 5% dextran wt/volume, with higher concentrations being without an effect on Ca²⁺ sensitivity.^20,24 Moreover, when dextran concentrations exceeded 10%, myofilament Ca²⁺ sensitivity may even be reduced.⁸ Hence, those data and our present data are consistent with the notion that the relation between interfilament spacing and myofilament Ca²⁺ sensitivity is highly nonlinear. This concept is illustrated in Figure 6 where Ca²⁺ sensitivity is plotted as function of myofilament lattice spacing. It is apparent that the effect of dextran treatment on Ca²⁺ sensitivity was limited to a narrow range of dextran concentrations, 1% to 3% under our conditions, whereas the myofilament lattice continued to shrink with increasing concentration of dextran in the solution. To differentiate whether the changes in Ca²⁺ sensitivity with dextran compression were the specific result of changes in lattice spacing, rather than some other, coincidental effect of osmotic compression, we performed a more critical test using 1% dextran. If interfilament spacing were the major determinant of crossbridge reactivity, then the Ca²⁺ sensitivity of moderately compressed trabeculae at the short SL should be similar to that of uncompressed trabeculae at the long SL given an equivalent lattice spacing. The data in Figures 5 and 6 show that this was not the case, again regardless whether pCa₅₀ or EC₅₀ was used to index Ca²⁺ sensitivity. Hence, our data are not consistent with the notion that changes in myofilament lattice spacing per se upon changes in SL within the physiological range are responsible for the increase in myofilament Ca²⁺ sensitivity in skinned cardiac muscle.

View larger version (15K): [in this window] [in a new window]   Figure 6. Relationship between myofilament lattice spacing and myofilament Ca2+ sensitivity in skinned cardiac trabeculae with varying concentrations of dextran (1% to 6%) in the solutions (n=7 to 10). Myofilament Ca2+ sensitivity is indexed by the EC50 parameter. Data are shown for a separate group of cardiac trabeculae at SL=2.19 µm () and SL=2.02 µm () in the absence of dextran. All other data are at SL=2.02 µm in the presence of dextran (closed symbols). Application of 1% dextran decreased myofilament lattice spacing equivalent to that of uncompressed trabeculae at 2.19 µm, without an increase in Ca2+ sensitivity (ie, no decrease in EC50). Dextran (3%) decreases lattice spacing and the EC50 (indicative of an increase in Ca2+ sensitivity). There is a further decrease in lattice spacing with 6% dextran without any further effect on Ca2+ sensitivity.

This conclusion differs from previous studies that provided support for the interfilament spacing hypothesis.^7,8,21 In those studies, however, changes in fiber width on either dextran compression or changes in SL were used to infer changes in myofilament lattice spacing. We found that the effect of different amounts of osmotic compression on muscle width is not equivalent to the effect on lattice spacing, as has been observed previously in skeletal muscle,¹⁵ albeit not uniformly.²² This is illustrated in Figure 7, where muscle width (as measured by microscopy) is plotted as a function of myofilament lattice spacing (as measured by x-ray diffraction) under conditions of varied SL and dextran concentration. The solid line (line of identity) shows the relationship that would be expected if muscle width were directly proportional to myofilament lattice spacing. These data show that application of 3% or 6% dextran reduced muscle width to a greater extent than myofilament lattice spacing. More importantly, SL change had a greater impact on muscle width than on myofilament lattice spacing. The deviation from proportionality increased with increases in dextran concentration. The mechanism that underlies these observations is unclear, but may result from a differential effect of dextran compression on muscle diameter and muscle width, such that compression caused the muscle to become more circular in cross-sectional shape. Alternatively, there may be a specific effect of dextran on the myofilament lattice alone, whereas changes in SL appear to affect all structures of the skinned trabecula, that is, both myofilament lattice spacing and extra-sarcomeric proteins. Nevertheless, under our conditions, to achieve a muscle width at the short SL equal to that at the long SL, approximately 2% to 3% dextran in the bathing solutions would have been required. Considering the nonlinear effect of dextran treatment on Ca²⁺ sensitivity (Figure 6), it is also apparent that 2% to 3% dextran treatment of skinned trabeculae would, in fact, be expected to significantly increase Ca²⁺ sensitivity, as indeed has been observed by the Moss group.⁷ By employing direct measurement of myofilament lattice spacing in skinned isolated cardiac trabeculae using x-ray diffraction, the confounding factor of using fiber width to estimate myofilament lattice spacing was eliminated in the present study.

View larger version (18K): [in this window] [in a new window]   Figure 7. Muscle width (as measured by microscopy) plotted as function of myofilament lattice spacing (as measured by x-ray diffraction) in skinned trabeculae (n=7 to 10). Muscle width and lattice spacing were determined at 3 sarcomere lengths (1.95 µm , 2.10 µm , and 2.25 µm ) with 0%, 3%, or 6% dextran in the solutions. The dotted line represents the line of identity between the 2 parameters (the predicted relationship if muscle width were directly proportional to myofilament lattice spacing; normalized to SL=2.10 µm; see text for details and discussion).

Why does Ca²⁺ sensitivity increase with application of dextran (Figures 1, 2, and 6; Table)? It may be argued that osmotic compression increases the number of attached crossbridges consistent with the increase in maximum force following dextran treatment. Yet, despite a significant increase in maximum force development on application of 1% dextran, there was no effect on myofilament Ca²⁺ sensitivity. Thus, osmotic compression at a short SL is not functionally equivalent to lengthening a sarcomere. Our data suggests that the effect of dextran treatment on Ca²⁺ sensitivity may be independent of changes in myofilament lattice spacing. Studies of skeletal muscle²⁵ suggest that there is a collapse of myosin heads against the myosin backbone over a range of osmotic pressures corresponding to about 1% to 3% dextran, ie, a structural effect of osmotic compression that is independent of lattice spacing. This could lead to the observed lack of change in Ca²⁺ sensitivity on increasing the dextran concentration above 3% because a decrease in crossbridge radius might be expected to counteract the reduction in lattice spacing. In principle, evidence for such a change in myosin structure could be obtained by analysis of the x-ray equatorial reflection intensities.²⁵ Whereas the minimal x-ray exposure times used to enhance sample lifetime in the present studies precluded such analysis, preliminary estimates of the 1,1 to 1,0 first order reflection intensity ratios in our study were consistent with previous findings in skeletal muscle.²⁵ Whether or not this mechanism underlies the increase in myofilament Ca²⁺ sensitivity on application of 3 to 6% dextran must await studies optimized for this purpose. Clearly, the possibility of structural changes in addition to the lattice spacing be must taken into account when interpreting any study in which dextran is employed to compress the myofilament lattice.

We have previously shown a similar dependence of interfilament spacing on sarcomere length in intact relaxed rat myocardium as compared with that in relaxed skinned trabeculae, albeit at a reduced interfilament spacing.¹¹ Our study does not directly address whether such a change in interfilament spacing underlies the length-dependent change in myofilament Ca²⁺ sensitivity in intact myocardium.²⁶ It has been observed that the effect of SL on Ca²⁺ sensitivity appears to be less in intact myocardium compared with skinned myocardium,²⁶ despite the similar impact of SL changes on interfilament spacing.¹¹ Hence, there appear to be differences between intact and skinned cardiac muscle, such that our results on skinned myocardium may not be applicable to intact heart. Nevertheless, if altered myofilament lattice spacing on a change in SL in skinned myocardium is not responsible for changes in Ca²⁺ sensitivity, as suggested here, what mechanism could account for length-dependent activation? It has been suggested that changes in Ca²⁺ sensitivity with length involve modulation of cooperative crossbridge binding to the thin filament.²⁷ Although sarcomere geometry and filament overlap may contribute to the availability of additional crossbridges along the ascending limb of the length-tension relationship,²⁸ it alone cannot account for the magnitude of the increase in force development.^3,7,29 It has been suggested that thin filament isoform proteins regulate the response to changes in length by some as of yet undetermined mechanism.⁵ Among alternative, plausible mechanisms that may be considered are those involving other sarcomeric proteins, such as the giant sarcomeric protein titin, which spans the entire sarcomere from Z-disk to Z-disk with interaction sites on both actin, myosin, and thin filament regulatory proteins.^30–33

In conclusion, we found that myofilament Ca²⁺ sensitivity did not vary in proportion to myofilament lattice spacing in osmotically compressed trabeculae. Furthermore, when the myofilament lattice was osmotically compressed so as to match the reduced lattice spacing as induced by increased sarcomere length, myofilament Ca²⁺ sensitivity was not significantly affected. These findings suggest that alterations in myofilament lattice spacing may not be the fundamental mechanism that underlies the sarcomere length–induced alteration of calcium sensitivity in skinned myocardium.

	Acknowledgments

 
This work was supported, in part, by the Cardiovascular Sciences Program Training Grant T32 07692 (J.P.K.), a national Grant-in-Aid from the American Heart Association 9950459N (T.C.I.), and NIH grants HL52322 and HL62426 (P.P.d.T.). Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Energy Research, under Contract No. W-31-109-ENG-38. BioCAT is a US NIH supported Research Center (RR08630). We thank Darold Perry, Dr Robert Fischetti, and Dr Karen Bischoff for assistance with the x-ray diffraction studies.

Received December 27, 2000; revision received October 22, 2001; accepted November 7, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Parmley WW, Chuck L. Length-dependent changes in myocardial contractile state. Am J Physiol. 1973; 224: 1195–1199.

2. Hibberd MG, Jewell BR. Calcium- and length-dependent force production in rat ventricular muscle. J Physiol. 1982; 329: 527–540.

3. Kentish JC, ter Keurs HE, Ricciardi L, Bucx JJ, Noble MI. Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle: influence of calcium concentrations on these relations. Circ Res. 1986; 58: 755–768.

4. Allen DG, Kurihara S. The effects of muscle length on intracellular calcium transients in mammalian cardiac muscle. J Physiol. 1982; 327: 79–94.

5. Arteaga GM, Palmiter KA, Leiden JM, Solaro RJ. Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I. J Physiol. 2000; 526: 541–549.

6. Godt RE, Maughan DW. Influence of osmotic compression on calcium activation and tension in skinned muscle fibers of the rabbit. Pflugers Arch. 1981; 391: 334–337.

7. McDonald KS, Moss RL. Osmotic compression of single cardiac myocytes eliminates the reduction in Ca²⁺ sensitivity of tension at short sarcomere length. Circ Res. 1995; 77: 199–205.

8. Wang YP, Fuchs F. Osmotic compression of skinned cardiac and skeletal muscle bundles: effects on force generation, Ca²⁺ sensitivity and Ca²⁺ binding. J Mol Cell Cardiol. 1995; 27: 1235–1244.

9. Rome E. X-ray diffraction studies of the filament lattice of striated muscle in various bathing media. J Mol Biol. 1968; 37: 331–344.

10. Matsubara I, Elliott GF. X-ray diffraction studies on skinned single fibres of frog skeletal muscle. J Mol Biol. 1972; 72: 657–669.

11. Irving TC, Konhilas JP, Perry D, Fischetti R, de Tombe PP. Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium. Am J Physiol. 2000; 279: H2568–H2573.

12. Harrison SM, Lamont C, Miller DJ. Hysteresis and length dependence of calcium sensitivity in chemically skinned rat cardiac muscle. J Physiol. 1988; 401: 115–143.

13. McDonald KS, Wolff MR, Moss RL. Sarcomere length dependence of the rate of tension redevelopment and submaximal tension in rat and rabbit skinned skeletal muscle fibres. J Physiol. 1997; 501: 607–621.

14. Fuchs F, Wang YP. Sarcomere length versus interfilament spacing as determinants of cardiac myofilament Ca²⁺ sensitivity and Ca²⁺ binding. J Mol Cell Cardiol. 1996; 28: 1375–1383.

15. Matsubara I, Goldman YE, Simmons RM. Changes in the lateral filament spacing of skinned muscle fibres when cross-bridges attach. J Mol Biol. 1984; 173: 15–33.

16. Konhilas JP, Irving TC, Smith SH, de Tombe PP. Osmotic compression and myofilament lattice spacing in rat cardiac trabeculae. Biophys J. 2001; 80: 1059.Abstract.

17. Gupta RC, Neumann J, Boknik P, Watanabe AM. M2-specific muscarinic cholinergic receptor-mediated inhibition of cardiac regulatory protein phosphorylation. Am J Physiol. 1994; 266: H1138–H1144.

18. Janssen PML, de Tombe PP. Protein kinase A does not alter unloaded velocity of sarcomere shortening in skinned rat cardiac trabeculae. Am J Physiol. 1997; 273: H2415–H2422.

19. Hammersley AP. FIT2DV9.129 Reference Manual Version 3.1. Grenoble, France: European Synchrotron Radiation Facility; 1998.

20. Zhao Y, Kawai M. The effect of lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers: II, elementary steps affected by the space change. Biophys J. 1993; 64: 197–210.

21. Fukuda N, Kajiwara H, Ishiwata S, Kurihara S. Effects of MgADP on length dependence of tension generation in skinned rat cardiac muscle. Circ Res. 2000; 86: e1–e6.

22. Kawai M, Wray JS, Zhao Y. The effect of lattice spacing change on cross-bridge kinetics in chemically skinned rabbit psoas muscle fibers: I, proportionality between the lattice spacing and the fiber width. Biophys J. 1993; 64: 187–196.

23. Lacampagne A, Cantos G, Vassort G. Is the regulation by stretch of the Ca²⁺ sensitivity of myofilaments in cardiomyocytes only controlled by the interfilament lattice spacing? J Muscle Res Cell Motil. 2000; 21: 818–819.Abstract.

24. Ford LE, Nakagawa K, Desper J, Seow CY. Effect of osmotic compression on the force-velocity properties of glycerinated rabbit skeletal muscle cells. J Gen Physiol. 1991; 97: 73–88.

25. Irving TC, Millman BM. Changes in thick filament structure during compression of the filament lattice in relaxed frog sartorius muscle. J Muscle Res Cell Motil. 1989; 10: 385–394.

26. Komukai K, Kurihara S. Length dependence of Ca²⁺-tension relationship in aequorin-injected ferret papillary muscles. Am J Physiol. 1997; 273: H1068–H1074.

27. Fitzsimons DP, Moss RL. Strong binding of myosin modulates length-dependent Ca²⁺ activation of rat ventricular myocytes. Circ Res. 1998; 83: 602–607.

28. Allen JD, Moss RL. Factors influencing the ascending limb of the sarcomere length-tension relationship in rabbit skinned muscle fibres. J Physiol. 1987; 390: 119–136.

29. ter Keurs HE, Rijnsburger WH, van Heuningen R, Nagelsmit MJ. Tension development and sarcomere length in rat cardiac trabeculae: evidence of length-dependent activation. Circ Res. 1980; 46: 703–714.

30. Labeit S, Kolmerer B, Linke WA. The giant protein titin: emerging roles in physiology and pathophysiology. Circ Res. 1997; 80: 290–294.

31. Cazorla O, Vassort G, Garnier D, Le Guennec JY. Length modulation of active force in rat cardiac myocytes: is titin the sensor? J Mol Cell Cardiol. 1999; 31: 1215–1227.

32. Cazorla O, Wu Y, Irving TC, Granzier H. Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes. Circ Res. 2001; 88: 1028–1035.

33. Coomber SJ, Tarasewicz E, Elliott GF. Calcium dependence of Donnan potentials in rigor: the effects of [Mg²⁺] and anions in isolated rabbit psoas muscle fibres. Cell Calcium. 1999; 25: 43–57.

This article has been cited by other articles:

				H. A. Shiels and E. White The Frank-Starling mechanism in vertebrate cardiac myocytes J. Exp. Biol., July 1, 2008; 211(13): 2005 - 2013. [Abstract] [Full Text] [PDF]

				L. M. Hanft, F. S. Korte, and K. S. McDonald Cardiac function and modulation of sarcomeric function by length Cardiovasc Res, March 1, 2008; 77(4): 627 - 636. [Abstract] [Full Text] [PDF]

				T. Terui, M. Sodnomtseren, D. Matsuba, J. Udaka, S. Ishiwata, I. Ohtsuki, S. Kurihara, and N. Fukuda Troponin and Titin Coordinately Regulate Length-dependent Activation in Skinned Porcine Ventricular Muscle J. Gen. Physiol., February 25, 2008; 131(3): 275 - 283. [Abstract] [Full Text] [PDF]

				K. Brixius, R. Lu, B. Boelck, S. Grafweg, F. Hoyer, C. Pott, U. Mehlhorn, W. Bloch, and R. H. G. Schwinger Chronic Treatment with Carvedilol Improves Ca2+-Dependent ATP Consumption in Triton X-Skinned Fiber Preparations of Human Myocardium J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 222 - 227. [Abstract] [Full Text] [PDF]

				T. E. Gillis, D. A. Martyn, A. J. Rivera, and M. Regnier Investigation of thin filament near-neighbour regulatory unit interactions during force development in skinned cardiac and skeleta muscle J. Physiol., April 15, 2007; 580(2): 561 - 576. [Abstract] [Full Text] [PDF]

				R. E. Petre, M. P. Quaile, E. I. Rossman, S. J. Ratcliffe, B. A. Bailey, S. R. Houser, and K. B. Margulies Sex-based differences in myocardial contractile reserve Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R810 - R818. [Abstract] [Full Text] [PDF]

				S. K. Frazier, K. S. Stone, D. Moser, R. Schlanger, C. Carle, L. Pender, J. Widener, and H. Brom Hemodynamic Changes During Discontinuation of Mechanical Ventilation in Medical Intensive Care Unit Patients Am. J. Crit. Care., November 1, 2006; 15(6): 580 - 593. [Abstract] [Full Text] [PDF]

				G. P. Farman, J. S. Walker, P. P. de Tombe, and T. C. Irving Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium Am J Physiol Heart Circ Physiol, October 1, 2006; 291(4): H1847 - H1855. [Abstract] [Full Text] [PDF]

				H. A. Shiels, S. C. Calaghan, and E. White The Cellular Basis for Enhanced Volume-modulated Cardiac Output in Fish Hearts J. Gen. Physiol., June 26, 2006; 128(1): 37 - 44. [Abstract] [Full Text] [PDF]

				J. T. Pearson, M. Shirai, H. Ito, N. Tokunaga, H. Tsuchimochi, N. Nishiura, D. O. Schwenke, H. Ishibashi-Ueda, R. Akiyama, H. Mori, et al. In Situ Measurements of Crossbridge Dynamics and Lattice Spacing in Rat Hearts by X-Ray Diffraction: Sensitivity to Regional Ischemia Circulation, June 22, 2004; 109(24): 2976 - 2979. [Abstract] [Full Text] [PDF]

				R. L. Moss, M. Razumova, and D. P. Fitzsimons Myosin Crossbridge Activation of Cardiac Thin Filaments: Implications for Myocardial Function in Health and Disease Circ. Res., May 28, 2004; 94(10): 1290 - 1300. [Abstract] [Full Text] [PDF]

				C. Levy and A. Landesberg Hystereses in the force-length relation and regulation of cross-bridge recruitment in tetanized rat trabeculae Am J Physiol Heart Circ Physiol, January 1, 2004; 286(1): H434 - H441. [Abstract] [Full Text] [PDF]

				N. Fukuda, Y. Wu, G. Farman, T. C Irving, and H. Granzier Titin isoform variance and length dependence of activation in skinned bovine cardiac muscle J. Physiol., November 15, 2003; 553(1): 147 - 154. [Abstract] [Full Text] [PDF]

				J. P Konhilas, T. C Irving, B. M Wolska, E. E Jweied, A. F Martin, R John Solaro, and P. P de Tombe Troponin I in the murine myocardium: influence on length-dependent activation and interfilament spacing J. Physiol., March 15, 2003; 547(3): 951 - 961. [Abstract] [Full Text] [PDF]

				G. M. Diffee and D. F. Nagle Exercise training alters length dependence of contractile properties in rat myocardium J Appl Physiol, March 1, 2003; 94(3): 1137 - 1144. [Abstract] [Full Text] [PDF]

				M. Helmes, C. C. Lim, R. Liao, A. Bharti, L. Cui, and D. B. Sawyer Titin Determines the Frank-Starling Relation in Early Diastole J. Gen. Physiol., February 3, 2003; 121(2): 97 - 110. [Abstract] [Full Text] [PDF]

				M. A. Sussman, A. McCulloch, and T. K. Borg Dance Band on the Titanic: Biomechanical Signaling in Cardiac Hypertrophy Circ. Res., November 15, 2002; 91(10): 888 - 898. [Abstract] [Full Text] [PDF]

				J. P Konhilas, T. C Irving, and P. P de Tombe Length-dependent activation in three striated muscle types of the rat J. Physiol., October 1, 2002; 544(1): 225 - 236. [Abstract] [Full Text] [PDF]

				H. Granzier and S. Labeit Cardiac titin: an adjustable multi-functional spring J. Physiol., June 1, 2002; 541(2): 335 - 342. [Abstract] [Full Text] [PDF]

				R. L. Moss and D. P. Fitzsimons Frank-Starling Relationship: Long on Importance, Short on Mechanism Circ. Res., January 11, 2002; 90(1): 11 - 13. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 90/1/59    most recent hh0102.102269v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Konhilas, J. P. Articles by de Tombe, P. P. Search for Related Content PubMed PubMed Citation Articles by Konhilas, J. P. Articles by de Tombe, P. P. Related Collections Structure Contractile function Calcium cycling/excitation-contraction coupling Cell biology/structural biology