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* Center for Cardiovascular Research, Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612-7342; and Department of Biological, Chemical, and Physical Sciences, Illinois Institute of Technology, Chicago, Illinois 60616
Correspondence: Address reprint requests and inquiries to Pieter P. de Tombe, E-mail pdetombe{at}uic.edu.
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ABSTRACT |
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 TOP ABSTRACT ACKNOWLEDGEMENTSREFERENCES |
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A unifying theory that has gained acceptance proposes that the impact of sarcomere length (SL) on myofilament Ca2+ sensitivity is due to changes in the spacing between the thick and thin filaments (1
,2). Because myofibrils are believed to maintain close to constant volume (3), elongation of the sarcomere is expected to lead to a reduction of the distance between the thick and thin filaments. A closer approximation of the myosin heads to actin may be expected to increase the probability of strong cross-bridge formation at a given concentration of activating calcium. Several experiments have provided support for this theory. Osmotic compression of the myofilament lattice by high molecular weight molecules that cannot enter the myofilament lattice structure, such as dextran, induces an increase in myofilament Ca2+ sensitivity concomitant with a reduction of muscle diameter (1–3). Since it has been shown that a reduction in muscle diameter occurs in parallel with a reduction in myofilament lattice spacing in skeletal muscle, the increase in myofilament Ca2+ sensitivity after dextran treatment without a change in SL is consistent with the interfilament spacing theory. Fuchs et al., who showed that myofilament Ca2+ sensitivity could be rendered length independent when muscle diameter was kept constant by applying an appropriate amount of dextran at each SL (1), provided more direct support for this theory. A similar result was obtained by McDonald et al., who showed in single permeabilized cardiac myocytes that application of dextran at a short SL was sufficient to increase myofilament Ca2+ sensitivity to match that recorded at the high SL, despite the fact that SL had not changed (2). On the other hand, we have recently suggested that interfilament spacing alone cannot be the only determinant of myofilament Ca2+ sensitivity (4,5). These conclusions, however, are based entirely on measurements of either muscle diameter or interfilament spacing in relaxed, noncontracting muscle preparations. It is possible that the myofilament lattice rearranges during contraction, the very condition that exists when myofilament Ca2+ sensitivity is assessed (3). Early experiments in skinned striated muscle suggested that upon activation there is an expansion of the myofilament lattice, which is likely due to the attachment of the myosin heads to the thin filament. A similar conclusion was also derived by Cecchi et al. (6), using a segment length clamp protocol in intact amphibian skeletal muscle. As in skeletal muscle, interfilament spacing in both intact and skinned myocardium is a function of SL (4), and large changes in SL usually accompany active contraction in isolated mammalian myocardium (7). Thus, whether systolic myofilament lattice spacing deviates from the diastolic lattice spacing in mammalian myocardium independent of changes in SL is currently unknown. Accordingly, to answer this question, we implemented an iterative feedback technique to control SL throughout the contraction in isolated rat cardiac trabeculae while interfilament spacing was simultaneously assessed by x-ray diffraction, either during diastole or at the peak of systole and over a wide range of SL and inotropic states.
All experiments were performed according to University of Illinois, Chicago, institutional guidelines concerning the care and use of experimental animals. Male rats (LBNF-1; 250-350 g) received intraperitoneal injections of 50 mg/kg sodium pentobarbital and 1.5 ml heparin (5). Under deep anesthesia, the heart was excised and perfused retrograde with a modified Krebs-Henseleit solution also containing 0.2 mM CaCl2 and 20 mM 2,3-butanedione monoxime to inhibit spontaneous contractions (25oC; pH 7.4) (5). Right ventricular trabeculae were dissected, mounted in the experimental apparatus, stimulated at 1 Hz, and left to equilibrate for 
30 min at [Ca2+] = 1.2 mM and diastolic SL = 2.1 µm. Next, three separate experimental runs were conducted at [Ca2+] = 0.3, 0.7 and 1.2 mM. In each run, SL was clamped by iterative feedback every 10th contraction at SL = 1.9, 2.0, 2.1, or 2.2 µm as previously described in detail (8). Briefly, SL clamp is achieved in the central segment of the muscle preparation close to the stationary attachment site of the muscle (i.e., the side attached to the force transducer) by appropriately stretching or releasing the muscle at the other side (connected to a high-speed motor). This approach allows for a SL clamp with minimal motion artifact at the site of measurement (8); an iterative feedback algorithm derives the muscle length profile that is required to achieve SL clamp. Briefly, the actual SL attained during the contraction (as measured by laser diffraction) is compared to the desired SL waveform so as to calculate a muscle length waveform via standard proportional integrative-differential feedback, which is to be applied during the next controlled contraction; this process is repeated over several contractions—usually 5–8—until the algorithm converges onto the desired SL waveform, after which several contractions can be recorded in succession under SL control (we averaged five contractions in this study). We carefully aligned a focused, shuttered x-ray beam (250 x 250 µm; 
= 0.103 nm; 10 ms) perpendicular to the laser beam and at the same area of the muscle where SL was controlled; this arrangement allows for the simultaneous measurement of myofilament lattice spacing and SL. Details of the x-ray apparatus have been described previously (5).
Fig. 1 A illustrates representative recordings obtained from a rat cardiac trabecula in a series of contractions with SL control from SL = 1.9 µm to 2.2 µm. In between the SL clamped contractions, diastolic SL was kept constant at SL = 2.1 µm such that each controlled contraction commenced at a comparable contractile state. For each SL controlled contraction, SL was stretched or released to the desired SL just before electrical stimulation and then held constant at that SL. Consistent with our previous studies (8), twitch force under SL clamp was both enhanced and prolonged as compared to SL auxotonic contractions (data not shown); furthermore, peak twitch force increased with increasing SL. The x-ray beam shutter was opened either during the peak of the contraction or just before stimulation in separate contractions under SL clamp as illustrated in panel C. The x-ray diffraction pattern was captured on a cooled charge-coupled device detector; diffraction patterns thus obtained either in diastole or peak systole allowed for accurate determination of interfilament spacing based on the d1,0 x-ray reflections. Fig. 1, panel D, illustrates the broadening as well as the shift in the ratio of the 1,0–1,1 intensity in going from diastole to systole, consistent with previous reports (3,6,9). It should be noted, however, that quantitative analysis of those aspects of the data would have required much longer x-ray exposures, and this was not the purpose of our experiments.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
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Submitted on January 12, 2007; accepted for publication January 25, 2007.
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2. McDonald, K. S., and R. L. Moss. 1995. Osmotic compression of single cardiac myocytes eliminates the reduction in Ca2+ sensitivity of tension at short sarcomere length. Circ. Res. 77:199–205.
3. Millman, B. M. 1998. The filament lattice of striated muscle. Physiol. Rev. 78:359–391.
4. Konhilas, J. P., T. C. Irving, and P. P. de Tombe. 2002. Frank-Starling law of the heart and the cellular mechanisms of length-dependent activation. Pflugers Arch. 445:305–310.[CrossRef][Medline]
5. Farman, G. P., J. S. Walker, P. P. de Tombe, and T. C. Irving. 2006. Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium. Am. J. Physiol. Heart Circ. Physiol. 291:H1847–H1855.
6. Cecchi, G., P. J. Griffiths, M. A. Bagni, C. C. Ashley, and Y. Maeda. 1991. Time-resolved changes in equatorial x-ray diffraction and stiffness during rise of tetanic tension in intact length-clamped single muscle fibers. Biophys. J. 59:1273–1283.
7. ter Keurs, H. E., W. H. Rijnsburger, R. van Heuningen, and M. J. Nagelsmit. 1980. Tension development and sarcomere length in rat cardiac trabeculae. Evidence of length-dependent activation. Circ. Res. 46:703–714.
8. Janssen, P. M., and P. P. de Tombe. 1997. Uncontrolled sarcomere shortening increases intracellular Ca2+ transient in rat cardiac trabeculae. Am. J. Physiol. Heart Circ. Physiol. 41:H1892–H1897.
9. Yagi, N., H. Okuyama, H. Toyota, J. Araki, J. Shimizu, G. Iribe, K. Nakamura, S. Mohri, K. Tsujioka, H. Suga, and F. Kajiya. 2004. Sarcomere-length dependence of lattice volume and radial mass transfer of myosin cross-bridges in rat papillary muscle. Pflugers Arch. 448:153–160.[CrossRef][Medline]
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), [Ca2+]o = 0.7 mM (
), and [Ca2+]o = 1.2 mM (
)). The dashed line indicates interfilament spacing as predicted by constant myofilament lattice volume. (B) Peak systolic interfilament spacing relative to diastolic interfilament spacing. Data presented as mean ± SE; n = 3.