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1Department of Physiology, University of Pennsylvania, School of Medicine, Philadelphia 19104-6085; and 2Department of Neurobiology and Anatomy, MCP-Hahnemann University, Philadelphia, Pennsylvania 19129
Submitted 4 October 2002 ; accepted in final form 28 April 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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thick filament structure; cardiomyopathy; filament stability
Rapid and reversible changes in thick filament structure and ordering of myosin heads can be produced in cardiac muscle by changes in the degree of phosphorylation of MyBP-C (22, 23), and these changes in structure are accompanied by changes in force production (11, 15). These interactions are probably the structural manifestation of the weak bonds formed before the entry of cross bridges into the force generating cycle. Major changes in contractility, including a large decrease in the maximum Ca-activated force (Fmax) and an increase in Ca sensitivity, can be produced by interference with the normal interactions of endogenous MyBP-C. The addition of certain fragments of MyBP-C or ligands of MyBP-C to skinned cardiac muscle can decrease Fmax by over 50% (1, 5, 10). The effect of these fragments on contractility depends on the state of phosphorylation of the added fragments. Thus under at least some conditions, cardiac MyBP-C seems to be necessary in the normal heart to permit normal force development. Further support for an important role of MyBP-C in cardiac muscle function is the fact that mutant forms of this protein can produce cardiac hypertrophy and dilation (2, 19, 21). Disruption of the myofibrillar structure and diminished power can be seen even at the stage when symptoms are mild or undetected (21).
Isolated cardiac myocytes fail to show any major impairment of contractility immediately after the extraction of a majority of MyBP-C (8). Extraction of 60–70% of MyBP-C does not cause any decrease in Fmax, although Ca sensitivity is increased. The increase in Ca sensitivity is reversed by the addition of MyBP-C to the bathing solution. These findings indicate that MyBP-C is not directly involved in the generation of maximum force and argue against a direct effect of MyBP-C on the individual force generating interactions between actin and myosin. It is possible that structural changes in the thick filament follow the removal of MyBP-C and alter the availability of force generators for entering the force generating cycle. For example, changes in the alignment of myosin heads with actin could modify the probability of actin and myosin interacting. The changes in structure may occur too slowly in an inactive bundle of skinned fibers to be unequivocally detected after 1 h of extraction.
The important question that remains is whether MyBP-C has a direct effect on the contractile proteins and their ability to generate force or an effect on the overall structure of the thick filament, which then influences the ability of the force generators to develop force. We have undertaken a study of the effect of extraction of MyBP-C on contractile performance of cardiac muscle and the structure of the thick filaments for a longer period after the onset of extraction. The results indicate that changes in thick filament structure and contractility occur after 4 h of extraction. They are either absent or difficult to detect after 1 h, and they can be reversed by the restoration of MyBP-C.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Extraction of MyBP-C. MyBP-C was extracted according to the protocol of Offer et al. (17) and of Hofmann et al. (8). The extraction solution consisted of 10 mM EDTA, 31 mM Na2HPO4, 124 mM NaH2PO4 adjusted to pH 5.9 with 3 µl/ml protease inhibitor cocktail (P-8340; Sigma), 1 µg/ml protein kinase inhibitors (type 3, P-0393; Sigma), and 1 µM okadaic acid, a phosphatase inhibitor. The relative amount of MyBP-C extracted was quantified by measuring the density of the band in Western blots after SDS-PAGE of the washout solutions and of the lysate of the muscle at the end of the experiment. The antibody was specific for the C0C1 modules of cardiac MyBP-C.
For extraction of MyBP-C, skinned trabeculae were first allowed to recover from the skinning procedure for 15–20 min in pCa of 9.0 (relaxing solution) and then exposed to pCa 6.0 to 4.5 as a control set of contractions. After a second 15–20 min period following the series of contractions, extraction of MyBP-C was begun and continued for 1 to 4 h in most cases. After several changes of relaxing solution to ensure complete removal of the extraction solution, trabeculae were bathed in a series of contraction solutions with pCa from 6.0 to 4.5 to determine the effect of extraction on Fmax and Ca sensitivity.
Preparation of MyBP-C and restoration to skinned fibers. MyBP-C was purified from hearts and kept at –80°C, according to procedures described in Hartzell and Glass (7) and Gautel et al. (4) with slight modifications. Myofibrils were purified by homogenizing isolated rat ventricles on ice at a concentration of 5 g tissue per 20 ml of buffer A [in mM: 50 KCl, 20 Tris-Cl, 2 EDTA, 15 2-mercaptoethanol, and "complete" protease inhibitors (Roche Molecular Biochemicals), pH 7.9] followed by spinning at 3,000 g for 15 min. The pellet was washed with buffer A four times and then 5 more times with buffer A containing 1% Triton. This step was followed by five more washes with buffer A.
The purified myofibrils were resuspended in 10 ml of buffer B (in mM: 10 EDTA, 300 KCl, 124 NaH2PO4 and 31 Na2HPO4, complete protease inhibitors, pH 5.9) and spun at 10,000 g for 20 min. This was repeated twice. Supernatants containing the C protein extracted from myofibrils were dialyzed against buffer C (same as buffer B, but with 50 mM KCl, pH 5.9), to precipitate myosin and then centrifuged at 10,000 g for 30 min. MyBP-C was then precipitated from this supernatant by ammonium sulfate at 45% saturation, and centrifuged at 10,000 g for 30 min. The pellet containing C protein was dissolved in buffer D (10 mM sodium phosphate buffer pH 7.0 with 300 mM KCl, complete protease inhibitors, and 1 mM EDTA).
MyBP-C was purified by three stages of chromatography: 1) fractionation on hydroxyapatite support by using CHT-II cartridge (Bio-Rad, Hercules, CA) according to instructions; 2) separation on Vivapure Spin Column (Vivascience) by using strong basic anion exchanger (Q), eluting C protein in 200 mM salt concentration; and 3) exchange of the existing buffer with the buffer required for experiment, immediately before the experiment gel filtration with D-Salt columns (Pierce, Rockford, IL). The purity of the MyBP-C preparation was checked by SDS-PAGE in which there was a single band migrating with the correct apparent molecular weight.
SDS-PAGE and Western blots for MyBP-C and myosin heavy chain. SDS-PAGE and Western blots were performed as described in McClellan et al. (15). A polyclonal antibody against C0C1 modules of MyBP-C and a monoclonal antibody against mouse slow skeletal myosin heavy chain (the latter obtained from Sigma, St. Louis, MO) were used. Details of the Western blotting with the antibody against C0C1 are given in McClellan et al. (15). For Western blotting with the monoclonal antibody against myosin, we used monoclonal antiskeletal myosin (slow) antibody (model M-8421; Sigma) as the primary antibody. Immunodetection was performed with the chemiluminescent system WesternBreeze-antimouse kit (Invitrogen, Carlsbad, CA).
The amount of MyBP-C and myosin heavy chain was measured absolutely and also normalized to the combined density of the actin and troponin T bands on the SDS gels. The normalized values were considered to be more reliable because of the possibility of some myofibrillar degradation in the course of the experiment. Amounts of protein added to each lane of the gel were restricted to those that produced staining within the linear range (15).
Electron microscopy and isolated thick filaments. Thick filaments were isolated, negatively stained, and viewed with transmission electron microscopy as described in Weisberg and Winegrad (22) and Levine et al. (11). Micrographs of individual thick filaments were subjected to optical diffraction, and the relative degree of order of myosin heads was quantified by comparing the intensity of the reflections along the 43-nm layer line as previously described (11).
The electron micrographs of several hundred filaments were subjected to optical diffraction as previously described (11, 22, 23). A mask around the image of the filament in the micrograph was used to eliminate interference from other filaments in the same micrograph and allow only one filament to produce the diffraction pattern. Because the mask itself can produce reflections along the meridian and the equator and some subjectivity may be involved in separating those from true thick filament reflections, only reflections off the meridian and along well-established myosin layer lines were used in judging thick filament structure. Reflections along the 43-nm layer line are produced by the helically arranged myosin heads and are the most useful in evaluating the degree of order of the myosin heads. This layer line normally has stronger reflections than the 14.3 layer line also produced by myosin heads (14). For this reason, the degree of order of myosin heads was evaluated by the relative intensity of the reflections along the 43-nm layer line in one quadrant of the optical diffraction pattern.
Up to three separate reflections along the 43-nm layer line were seen with ordered filaments. Each optical diffraction pattern was digitized and the relative intensity along the 43-nm layer line was determined by the National Institutes of Health (NIH) Image program. The area times the intensity of each reflection was measured, and the values were added together to give the total reflection.
The relative intensity of specific reflections in an optical diffraction pattern was determined by a direct comparison among optical diffraction patterns from different micrographs. A second method, in which the intensity of the reflection was compared with the average intensity of the reflections along the meridian between 43 and 14 nm to provide an internal standard in each film, gave similar results as long as the exposure and development times were the same.
Statistics. Values for forces were expressed as means ± SE.
Student's t-test was used to determine significance. Scans of
diffraction patterns and gels were made with the NIH Image program.
Distributions of the populations of filaments were tested for normality by
using the Lilliefors test with the SPSS Graduate pack. Differences in
variances were sufficiently small to support the use of the t-test.
Distributions of intensity of reflections in the optical diffraction patterns
were fit by equations for two normal distributions with Origin software
(Microcal Software), and the goodness of fit was measured by the
2-test. Values were considered statistically significantly
different when P < 0.05. Where multiple comparisons were made, the
P value was corrected by using the Bonferroni method.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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To be able to attribute the changes in contractility of the cardiac cells
to the loss of MyBP-C, it was essential to rule out the possibility that other
myofibrillar proteins were lost or altered during the extraction of MyBP-C.
With the use of SDS polyacrylamide electrophoresis, we carefully examined
highly concentrated aliquots of the extraction solutions in every experiment
in which MyBP-C was removed. MyBP-C, confirmed by Western blotting with an
antibody specific for the C0C1 portion of cardiac
MyBP-C, was present with, at most, only a trace of any other protein
(Fig. 1). Occasionally, there
were two additional bands at 
34 kDa, but Western blotting indicated that
these bands were proteolytic fragments of MyBP-C. No significant loss of
myosin heavy chain or myosin light chains occurred (3 ± 1%) during 4 h
of extraction of MyBP-C, nor were thin filament proteins detected in the
extraction solution. Because loss of troponin decreases Ca regulation of
contraction and alters baseline tension, a second test for loss of troponin
was the degree to which the extraction of MyBP-C changed baseline force.
During extraction and for at least 4 h afterward, no change in baseline
tension occurred in any experiment. These results indicate that any change in
Fmax during the first 4 h of extraction was produced by loss of
MyBP-C and not other myofibrillar proteins.
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The relative amount of MyBP-C extracted was determined from the amounts of MyBP-C in the extraction solutions and remaining in the trabeculae. Because the intensity of staining was compared for the same protein in the same gel, difference in sensitivity to gel stains was not a problem. During the first hour of extraction, 18 ± 3% of the tissue content of MyBP-C was extracted and after 4 h the percentage of extracted MyBP-C increased to 29 ± 3% (Fig. 2, Table 1).
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Extraction of MyBP-C for 1 h produced an increase in Ca sensitivity and a small but statistically insignificant decline in Fmax as seen by Hofmann et al. (8) (Fig. 3). After 4 h of extraction of MyBP-C, which removed 29 ± 3% of the protein, Fmax declined to 74% its original value, and the trabeculae became more sensitive to Ca (Fig. 3).
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Reversibility of the decline in Fmax with added MyBP-C. To determine whether the changes in Fmax and Ca sensitivity were due specifically to the removal of MyBP-C, extracted trabeculae were incubated for 1 h in a relaxing solution containing 3 µM MyBP-C that had been purified from a rat heart. The purified MyBP-C normally produced a single band (Fig. 4B, lane 4), and only a trace of another band when the lane was heavily loaded (Fig. 4B, lane 3) on SDS-PAGE gels. After 1-h incubation, the solution was changed to relaxing solution for 30 min to wash out the unbound MyBP-C, and then the trabeculae were exposed to Ca containing solutions.
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Results of the incubation with MyBP-C fell into two groups; those in which restoration of Fmax was complete or nearly complete and those in which little or no recovery was produced by the exposure to MyBP-C (Table 1). In 10 of 14 experiments, Fmax and Ca sensitivity were successfully restored to their original values (Figs. 3 and 4). Fmax after exposure to MyBP-C in the 10 experiments was 94 ± 4% of the control. The addition of an equivalent amount of BSA had no effect. In the second group, Fmax or the change in Ca sensitivity after exposure to MyBP-C was 76 ± 5%. The mean for all 14 experiments was 89 ± 4%. There was no difference in the magnitude of the decline in Fmax after extraction of MyBP-C between the two groups. The differences of Fmax and Ca sensitivity between the 4-h MyBP-C extracted and the 1-h MyBP-C restored trabeculae were statistically significant when all 14 experiments were analyzed together (Table 1; P < 0.05).
Effect of loss of MyBP-C during quiescence. A second method of reducing the MyBP-C content of heart muscle does not require exposure of the tissue to low pH or a high concentration of phosphate. MyBP-C is lost from intact trabeculae that do not contract during both incubation for 2–3 h in a standard medium before skinning and an additional 2 h in relaxing solution without Ca activation after skinning. These trabeculae lost 12 ± 3% of their MyBP-C to the bathing solutions (n = 11; P < 0.01) (Fig. 5). With this protocol, it is not possible to compare contractility in the same tissue before extraction with that after restoration of MyBP-C because activation of contraction prevents the loss of MyBP-C. The problem can be overcome by using paired preparations. One muscle was activated to contract by Ca 15 min after the completion of skinning. Muscles that contracted did not lose MyBP-C, and they produced 27 ± 2 mN/mm2. The other member of the pair was not activated to contract by Ca for 2 h after skinning, by which time Fmax had decreased to 19 ± 2 mN/mm2 (n = 11; P < 0.02).
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After measuring Fmax in trabeculae partially depleted of MyBP-C in this way, we incubated them in relaxing solution containing 3 µM MyBP-C either for 1 h at room temperature or overnight at 4 C. In the latter case, the muscles were brought to room temperature in relaxing solution without MyBP-C. After the change to a relaxing solution without MyBP-C, the contractile response to increasing Ca was again measured. Fmax of the quiescent muscles in each pair had increased by 31 ± 4% (n = 11; P < 0.01) (Fig. 6). pCa for 50% of Fmax had decreased by 0.14 ± 0.06 pCa units (P < 0.05). These changes were the opposite of those produced by removing MyBP-C and did not occur when MyBP-C was not in the overnight bathing solution nor did they occur when an equal concentration of BSA was present instead of MyBP-C (data not shown). Increase in Fmax and decrease in Ca sensitivity were maintained for at least 12 h. Incubation in MyBP-C of the paired muscles that had been Ca activated and had not lost MyBP-C, caused a small but statistically insignificant increase in Fmax.
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Effect of extraction of MyBP-C on structure of thick filament. Thick filaments isolated from cardiac muscle have ordered or disordered myosin heads depending on the protocol before the isolation of the filaments (11, 22). Most thick filaments from heart muscle soaked in Krebs solution containing 2.5 mM Ca and activated shortly after skinning have an ordered structure with myosin heads lying along the surface of the filament at a regular periodicity (Fig. 7). Optical diffraction of micrographs of these filaments produces strong reflections along the 43-nm layer line (11, 15). A minor fraction of the filaments have visibly disordered myosin heads and a very low intensity of reflections along the 43-nm layer line. Filaments retain their structure for at least 24 h.
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After4hof extraction of MyBP-C by Offer's solution, most of the thick filaments isolated from cardiac muscle were disordered and had myosin heads extending at a variety of angles from the backbone of the filament (Fig. 7). The relative intensity of the reflections along the 43-nm layer line produced by micrographs of these filaments had peaks at similar intensities to control unextracted filaments, but the fraction of filaments in each peak was different (Fig. 7). Micrographs of a majority of filaments from extracted muscle produced weak or no reflections along the 43-nm layer line. Difference in distributions of filaments between the two populations in control and extracted tissue was highly significant (P < 0.01).
We examined the distribution of intensity of reflections produced by micrographs of filaments from muscles in which incubation with 3 µM MyBP-C had restored Fmax. The distribution after incubation was different from that before incubation with MyBP-C (P < 0.02) (Fig. 7) and resembled that of the control. There was no significant difference between the control and the MyBP-C restored distributions. No significant difference in the distribution of intensity was produced by incubation with MyBP-C when there was no change in Fmax.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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One hour of exposure to extraction solution removed 18% of the MyBP-C from skinned trabeculae without changing Fmax. Removal of 60–70% of MyBP-C from isolated, mechanically disrupted single cardiac myocytes over the same period of time produced a similar effect on contractility: no change in Fmax and an increase in Ca sensitivity. When the extraction from trabeculae was continued for an additional 3 h, a total of 29% of MyBP-C was removed, and further changes in contractility occurred. Fmax decreased by 25%, and the contractile system became more sensitive to Ca without any detectable loss of myosin or thin filament proteins such as actin or troponin. The decline in Fmax that occurred with extraction of MyBP-C for 4 h was completely reversed in a majority of preparations by incubation in a relaxing solution containing purified MyBP-C. This is strong evidence that the removal of MyBP-C was responsible for the decline in contractility produced by the soak in Offer's solution.
There are two possible general explanations for the different results after 4 h of extraction of MyBP-C from chemically skinned cardiac trabeculae compared with results after 1 h with isolated, mechanically disrupted cardiac myocytes. Either processes affecting contractility were initiated during 1 h of extraction of MyBP-C, but were not detectable by measurement of Fmax until they had evolved further, or the changes in contractility required a larger loss of MyBP-C from the skinned trabeculae than occurs in 1 h. The latter is the less likely explanation. Removal of 60–70% of MyBP-C over 1 h from mechanically disrupted myocytes produced similar changes to extraction of 18% of MyBP-C for 1 h from skinned trabeculae: reversible increase in Ca sensitivity and unchanged Fmax. However, extraction of 29% from skinned trabeculae over 4 h decreased Fmax. These effects on Fmax and Ca sensitivity were reversed in a majority of preparations by incubating the tissue in relaxing solution containing rat cardiac MyBP-C.
During a prolonged period without contractile activity before and after
skinning, a portion of MyBP-C was lost from the skinned trabeculae. Associated
with this loss was a decline in Fmax estimated to be 30% on
the basis of comparison with paired trabeculae that had not been maintained
quiescent. With this protocol, loss of MyBP-C occurred without exposure to low
pH or high phosphate-containing solutions. MyBP-C added to the bathing
solution reversed the decline in Fmax. Because a minor fraction of
MyBP-C was involved, it was not possible to make sufficiently precise
measurements of the amount of MyBP-C taken up by the muscle during the
incubation in MyBP-C. The effect of loss of MyBP-C by prolonged quiescence
before and after skinning is qualitatively similar to the effect of extraction
by Offer's solution: decline in Fmax and increase in Ca sensitivity
reversed in both preparations by incubation with MyBP-C. The fact that removal
of an approximately equal amount of MyBP-C in a much shorter time (1 h) by
Offer's solution produced no decline in Fmax supports the inference
that a slow, time-dependent change in thick filament is involved in the change
in Fmax.
There are several possible reasons why the rate of extraction of MyBP-C was substantially quicker in the study of Hofmann et al. (8) than in our work. The preparations are quite different: theirs is a single cell isolated by homogenization of cardiac muscle followed by exposure to 3% Triton X-100, and ours is an isolated thin trabecula chemically skinned with 1% Triton X-100. The diffusion distances were much smaller in the isolated myocyte than the skinned trabecula.
The structure of the thick filament changes in parallel with the extraction and replacement of MyBP-C. Before extraction, the myosin heads in a majority of thick filaments were well ordered. After extraction for 4 h with Offer's solution, the majority of thick filaments had disordered myosin heads. Order was restored after incubation with MyBP-C when that incubation raised Fmax. Thick filaments with disordered myosin heads have been associated with low Fmax in other conditions, particularly in the presence of a reduced level of phosphorylation of MyBP-C (11, 15). These correlations suggest that the mechanism by which Fmax is reduced with extraction of MyBP-C is a change in the structure of the thick filament that affects the relationship of the myosin heads to the backbone of the filament and presumably to the interactive sites on actin in the thin filament. Such a mechanism is consistent with the fact that although there is only one molecule of MyBP-C for each seven molecules of myosin, changes in MyBP-C can alter maximum force generation by over 50% (10, 15). It is likely that MyBP-C exerts at least part of its effect on contractility by altering overall structure of thick filaments rather than direct interactions with contractile proteins.
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DISCLOSURES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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