AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 283: H642-H649, 2002. First published April 25, 2002; doi:10.1152/ajpheart.00890.2001
0363-6135/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/2/H642    most recent
00890.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (7)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Martin, A. F.
Right arrow Articles by Solaro, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Martin, A. F.
Right arrow Articles by Solaro, R. J.
Vol. 283, Issue 2, H642-H649, August 2002

Ca2+ activation and tension cost in myofilaments from mouse hearts ectopically expressing enteric gamma -actin

Anne F. Martin1,*, Ronald M. Phillips1,*, Ajit Kumar2, Kelly Crawford2, Zainab Abbas2, James L. Lessard2, Pieter de Tombe1, and R. John Solaro1

1 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612; and 2 Children's Hospital Research Foundation, Cincinnati, Ohio 45229


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine the significance of actin isoforms in chemomechanical coupling, we compared tension and ATPase rate in heart myofilaments from nontransgenic (NTG) and transgenic (TG) mice in which enteric gamma -actin replaced >95% of the cardiac alpha -actin. Enteric gamma -actin was expressed against three backgrounds: mice expressing cardiac alpha -actin, heterozygous null cardiac alpha -actin mice, and homozygous null cardiac alpha -actin mice. There were no differences in maximum Ca2+ activated tension or maximum rate of tension redevelopment after a quick release and rapid restretch protocol between TG and NTG skinned fiber bundles. However, compared with NTG controls, Ca2+ sensitivity of tension was significantly decreased and economy of tension development was significantly increased in myofilaments from all TG hearts. Shifts in myosin isoform population could not fully account for this increase in the economy of force production of TG myofilaments. Our results indicate that an exchange of cardiac alpha -actin with an actin isoform differing in only five amino acids has a significant impact on both Ca2+ regulation of cardiac myofilaments and the cross-bridge cycling rate.

cross bridge; myosin isoforms; energy coupling


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE QUESTION OF WHETHER THE small amino acid differences between muscle-specific isoforms of actin influence myofilament activation and chemomechanical coupling has remained elusive. One problem is a lack of measurements comparing myofilaments with specific differences in actin isoforms within the framework of the myofilament lattice. A second problem is that muscle isoforms of actin appear to be interchangeable in in vitro assays of muscle function. For example, Harris and Warshaw (15) reported that the sliding behavior of filaments composed of either smooth or skeletal actins in the motility assay is indistinguishable. Moreover, Korman and Tobacman (20), who used mutations in yeast actin to analyze structure-function relations that alter thin filament regulation by tropomyosin and troponin, suggest that differences in homology between yeast and striated muscle actins have little impact on actin function in vitro. Yet ectopic expression of beta - or gamma -cytoplasmic actins in isolated neonatal or adult cardiomyocytes caused significant alterations in cellular morphology, including disassembly of myofibrillar thin filaments and inhibition of contraction (41). However, expression of alpha -skeletal, alpha -smooth muscle, or enteric gamma -actin in cardiomyocytes had very little effect on myofibrillar structure, thin filament assembly or ability to contract (41).

In contrast to in vitro studies, functional differences associated with the expression of different isoforms of actin are seen in studies of whole hearts. Perfused hearts isolated from BALB/c mice, which naturally express high levels of skeletal alpha -actin (10), are hyperdynamic and show increased levels of contractility (16). On the other hand, in cardiac alpha -actin-deficient mouse hearts that are rescued by targeted ectopic expression of enteric smooth muscle gamma -actin in the cardiac compartment, the hearts are hypodynamic (21). Both these observations support the concept that a few amino acid differences between muscle actin isoforms can have functional consequences. Moreover, the discovery that point mutations in cardiac actin are linked to heritable forms of both dilated (30) and hypertrophic (29) cardiomyopathies strongly indicates that minor localized changes in actin may be amplified into major functional abnormalities in the whole heart.

In experiments reported here, we tested whether the amino acid differences between cardiac alpha -actin and enteric gamma -actin affect activation and chemomechanical coupling of cardiac myofilaments. Our approach involved the study of myofilaments isolated from hearts of transgenic (TG) mice expressing enteric smooth muscle gamma -actin. Compared with myofilaments from control mice, myofilaments containing enteric gamma -actin demonstrated a decreased sensitivity to Ca2+ and an increased economy of force development. Our results indicate that the five amino acid differences between cardiac and enteric gamma -actin (Table 1) affect its interaction with both troponin and myosin cross-bridges.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Amino acid differences between cardiac alpha -actin and enteric gamma -actin


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal models. TG mice that ectopically express enteric gamma -actin in the heart were generated as previously described (21). Enteric gamma -actin was expressed against three backgrounds: 1) wild-type (WT) mice expressing cardiac alpha -actin (+/+), 2) heterozygous null cardiac alpha -actin mice (+/-), and 3) homozygous null cardiac alpha -actin mice (-/-). Most mice lacking cardiac alpha -actin (-/-) die within 2 wk after birth. Nontransgenic (NTG) WT littermates (+/+) and NTG heterozygous cardiac alpha -actin (+/-) littermates were used as control groups. Data from these two control groups were combined into a single group of NTG because we observed no statistical differences between these two groups of NTG mice. Some NTG mice from both (+/+) and (+/-) backgrounds were placed on a diet containing 0.15% propylthiouracil (PTU) obtained from Research Diets (Madison, WI) for time periods ranging from 1 to 8 wk as indicated.

Quantification of actin isoforms expressed in heart. Myofibrils were isolated from adult ventricle, and solubilized in sodium dodecyl sulfate (SDS) sample buffer, and the proteins present were separated by denaturing 12% polyacrylamide gel electrophoresis (PAGE), as described by Laemmli (22). After electrophoresis, triplicate samples of myofibrils were electroblotted onto nitrocellulose (39). Purified cardiac alpha -actin and purified chicken gizzard (enteric) gamma -actin were run on gels in parallel to myofibrillar samples to generate standard curves (63-500 ng actin) and to act as internal controls. Total actin present was determined using monoclonal antibody (mAb) C4 (25). Striated actins, both cardiac and skeletal alpha -actins, were detected using mAb 5C5 (34) and enteric gamma -actin with mAbB4 (25). The binding of monoclonal antibodies to actin isoforms was quantified using the Vectastain ABC system (Vector Laboratories; Burlingame, CA). The immunoblots were scanned and the digitized images were analyzed with the use of NIH Image software.

Preparation of fiber bundles. Adult mice of either sex were anesthetized with pentobarbital sodium (50 mg/kg ip), and the hearts were rapidly excised, rinsed in ice-cold saline, and placed in ice-cold relaxing solution (pH 7.0) composed of (in mM) 79.2 KCl, 6.5 MgCl2, 5.4 Na-ATP, 10 EGTA, 30.0 3-(N-morpholino)propanesulfonic acid, and 12 creatine phosphate. This solution also contained 10 U/ml creatine phosphokinase and the following protease inhibitors: 1 µg/ml leupeptin, 2.5 µg/ml pepstatin A, and 50 µM phenylmethylsulfonyl fluoride. The papillary muscles from the left ventricle were dissected free and small fiber bundles (~150-250 µm in diameter and 1-3 mm in length) were prepared as previously described (44). The fiber bundles were extracted overnight in relaxing solution plus 1% (vol/vol) Triton X-100 at 4°C.

Simultaneous determination of force and ATPase activity. Force and ATPase rate were measured simultaneously as described previously (44) using an experimental apparatus previously described in detail by de Tombe and Stienen (7). The fiber bundles were mounted between a force transducer (model AE801, SensoNor) and displacement motor (model 300B, Cambridge Technology) using aluminum T-clips. The sarcomere length was set at 2.15 µm, as determined using He-Ne laser diffraction (6). Width and diameter were measured at three points along the fiber bundle and the cross-sectional area was estimated based on an elliptical model. Tension was computed as force per cross-sectional area.

ATPase activity (at 20°C) was measured in an enzyme- coupled spectrophotometric assay at 340 nm in which generation of ADP was stoichiometrically coupled to the conversion of reduced nicotinamide adenine dinuncleotide to nicotinamide adenine dinuncleotide by pyruvate kinase and lactate dehydrogenase. The reaction was carried out in a 25-µl cuvette in solutions described previously (44). Calibration was performed by rapid injection of ADP (0.5 nmol) with a motor-controlled syringe after a contraction-relaxation cycle. During each series of measurements, the fiber bundle was incubated in the relaxing solution for 4 min, in the preactivating solution for 3 min, in the activating solution for ~2 min, and then again in relaxing solution. Before the first activation-relaxation cycle, sarcomere length was adjusted to 2.15 µm in relaxing solution. After an initial contraction at saturating Ca2+ concentration ([Ca2+]) (50 µM), sarcomere length was then readjusted to 2.15 µm. At this point, resting sarcomere length remained stable throughout the experiment (7). The next five to six contraction-relaxation cycles were carried out at a range of intermediate [Ca2+] generated by varying the ratio of total [Ca2+] to total EGTA concentration. Finally, another contraction at saturating Ca2+ was recorded. A computer program was used to calculate the amount of CaCl2 required to generate a range of free [Ca2+] (9, 13). The tension cost was calculated from the slope of the plot between tension and ATPase activity measured simultaneously in individual skinned fibers over a range of [Ca2+] and ATPase activities including maximal ATPase.

Measurement of rate of tension redevelopment and stiffness. The slack/release approach described by Brenner and Eisenberg (5) was used to disengage force-generating cross bridges from isometrically activated thin filaments. Sarcomere length was set at 2.15 µm. We activated the fiber bundles by rapidly transferring them from the preactivation solution containing a high concentration of EGTA to activating solutions with varying free Ca2+. Once the steady-state level was achieved, force was decreased to 0 within 1 ms by imposing a slack equivalent to 10% of the muscle length, followed immediately by unloaded shortening for 20 ms. The remaining bound cross bridges were mechanically detached by rapidly (1 ms) restretching the muscle fiber to its original length. The tension redevelopment data were fitted to a monoexponential function. The activation dependence was determined by measurements at saturating Ca2+ (50 µM) and two other [Ca2+]. Fiber stiffness was determined by applying length perturbations (1% muscle length). The resultant force response at 500 Hz was measured by means of a dual-phase lock-in amplifier (Stanford Instruments). Fiber stiffness was calculated as the ratio of change in force to change in muscle length.

Measurement of alpha -to-beta -myosin heavy chain ratios. The alpha - and beta -myosin heavy chains (MHC) present in fiber bundles were determined by electrophoresis on 6% polyacrylamide gels as described by Laemmli (22) with the following modifications. The polyacrylamide-to-bis-acrylamide ratio was 100:1 and the temperature of the running buffer was maintained between 5 and 6°C during electrophoresis. The fiber bundles were solubilized in a sample buffer consisting of 50 mM Tris, pH 6.8, 2.5% SDS, 10% glycerol, 1 mM dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml aprotinin. Two to three fiber bundles from a single heart were combined to run on the gel. In some cases, left ventricular tissue was pulverized in liquid nitrogen and extracted in sample buffer, and 5-10 µg of protein were loaded per lane. Gels were stained with Coomassie blue (Pierce Gelcode) and the relative amounts of alpha - and beta -MHC determined by densitometric analysis by using a Molecular Dynamics SI densitometer. The areas under the peaks were quantified using QuantImage Software.

Data analysis. Data are expressed as means ± SE. Statistical significance was verified using one-way analysis of variance, followed by a Newman-Keuls multiple-comparison post hoc test. P <=  0.05 was considered significant. The relation between Ca2+ and tension or ATPase activity was fitted by nonlinear least-squares regression to a modified Hill equation
P = max [Ca<SUP>2+</SUP>]<SUP><IT>n</IT></SUP>/([Ca<SUP>2+</SUP>]<SUP><IT>n</IT></SUP> + EC<SUP><IT>n</IT></SUP><SUB>50</SUB>)
where P is tension or ATPase activity, max is the maximum value at 50 µM Ca2+, EC50 is [Ca2+] at half-maximal activation, and n is the Hill coefficient. The relation between beta -MHC and economy of force production was analyzed by multiple linear regression analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of enteric gamma -actin in the heart. The isotypes of actin expressed in ventricular tissue from NTG and TG mice ectopically expressing enteric gamma -actin were determined using antibodies reacting with all actin isotypes (mAbC4), striated actins both skeletal and cardiac (mAb5C5), and enteric gamma -actin (mAbB4) (see Table 2). Total actin expressed in NTG (+/+) or (+/-) groups consisted of 60% striated actin isoforms and negligible amounts of enteric gamma -actin. In hearts from TG (+/+) mice, expression of striated actins was reduced to 16% of the total actin and enteric gamma -actin comprised 85.6% of the total actin population. TG (+/-) mouse hearts contained 76.7% enteric gamma -actin and 5% striated actins. In TG (-/-) mice, essentially all of the actin expressed in the heart was enteric gamma -actin. Thus, in all three TG groups ectopically expressing enteric gamma -actin in the cardiac compartment, the vast majority of the actin expressed in the ventricle consisted of enteric gamma -actin. Table 1 summarizes the amino acid differences between cardiac alpha -actin and enteric gamma -actin.

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Expression of striated and enteric actins in myofibrils from NTG and enteric gamma -actin TG mouse hearts

Analysis of total extracts of ventricular tissue from NTG and TG mice by PAGE on 12.5% gels showed no obvious differences in protein profiles. Densitometric analysis of the Coomassie blue-stained gels (Molecular Dynamics SI densitometer) and quantification of the MHC and actin bands with the use of ImageQuant software indicated no significant differences between the NTG and TG samples. The MHC-to-actin ratio in hearts from NTG mice was 0.83 ± 0.07 (n = 3) and 0.86 ± 0.04 (n = 7), respectively, in hearts from TG animals.

Tension, stiffness, and maximal rate of tension redevelopment of skinned fiber bundles. The Ca2+ sensitivity of skinned fiber bundles from TG mouse hearts ectopically expressing enteric gamma -actin was significantly decreased as indicated by a rightward shift of the [Ca2+]-tension relationship compared with NTG littermates expressing cardiac alpha -actin (Fig. 1 and Table 3). The [Ca2+] required for the development of EC50 was significantly higher in all three TG groups (+/+, +/-, and -/-) compared with NTG mice (Table 3). Both TG (+/+) and (-/-) showed an increase in apparent cooperativity (Hill coefficient), although the apparent cooperativity of the TG (+/-) mice did not differ from that of the NTG group. We observed no consistent changes in maximum tension between fibers containing enteric gamma -actin and those containing only cardiac alpha -actin (Table 3). Fiber bundles from hearts expressing enteric gamma -actin on the WT (+/+) background, however, did appear to generate significantly more force than NTG controls.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1.   Ca2+-isometric tension relation in detergent-treated cardiac fiber bundles from nontransgenic (NTG) and enteric gamma -actin transgenic (TG) mice. Measurements were made as described in MATERIALS AND METHODS. Data are presented as means ± SE. NTG, n = 6 (16); TG +/+, n = 3 (8); TG +/-, n = 3 (6); TG -/-, n = 5 (10). Numbers in parentheses are the number of fiber bundles.


                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Ca2+ sensitivity of tension in fiber bundles from NTG and enteric gamma -actin TG mice

Measurements of the relation between fiber stiffness and Ca2+-activated tension indicated that force produced per cross-bridge was not altered in fiber bundles containing enteric gamma -actin (Fig. 2). Data illustrated in Fig. 3 also indicate that the maximal rate of tension redevelopment (Ktr) was not significantly affected by substitution of enteric gamma -actin for cardiac alpha -actin in the cardiac myofilaments.


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2.   Comparison of the slope of the stiffness-tension relation at a series of Ca2+ concentrations in cardiac fiber bundles from NTG and enteric gamma -actin TG mice. Experimental conditions are described in MATERIALS AND METHODS. Data are presented as means ± SE. NTG, n = 6 (15); TG +/+, n = 3 (8); TG +/-, n = 3 (6); TG -/-, n = 5 (9). Numbers in parentheses are the number of fiber bundles analyzed.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 3.   Maximal rate of tension redevelopment (Ktr) in cardiac fiber bundles from NTG and enteric gamma -actin TG mice. Experimental procedures are described in MATERIALS AND METHODS. Maximal Ktr was measured at 50 µM Ca2+. Data are presented as means ± SE. NTG, n = 8 (13); TG +/+, n = 4 (7); TG +/-, n = 3 (6); TG -/-, n = 4 (7). Numbers in parentheses are the number of fiber bundles analyzed.

Tension cost. We simultaneously measured tension and ATPase activity in fiber bundles containing cardiac alpha -actin or enteric gamma -actin to evaluate possible differences in the energy cost for the development of unit force. As shown in Fig. 4, the tension cost in fiber bundles from all three groups of TG mice was significantly reduced when compared with fiber bundles from NTG control mice. About 35% less ATP hydrolysis was required to maintain a given level of tension in fiber bundles containing enteric gamma -actin than in fiber bundles containing cardiac alpha -actin.


View larger version (9K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of expression of enteric gamma -actin in mouse heart on the tension cost in fiber bundles from TG and NTG mice. Data represent the slope of the ATPase-tension relation measured at several Ca2+ concentrations ([Ca2+]). Experimental conditions are given in MATERIALS AND METHODS. The data are presented as means ± SE. NTG, n = 8 (15); TG +/+, n = 4 (8); TG +/-, n = 3 (6); TG -/-, n = 5 (10). Numbers in parentheses are the number of fiber bundles analyzed. *P <=  0.001 compared with NTG.

The possibility that altered expression of the myosin isoform population was responsible for the decrease in tension cost observed in fiber bundles from TG mice was investigated. The maximal rate of ATPase activity in a variety of muscles has been directly related to the isoform of MHC present (2). Two isoforms of MHC, alpha  and beta , are expressed in cardiac tissue and give rise to the myosin isoforms V1 (alpha alpha ), V2 (alpha beta ), and V3 (beta beta ) (17). V1 has a two- to threefold faster rate of ATP hydrolysis than the V3 isoform and also a higher tension cost (1, 27). Electrophoretic separation of alpha - and beta -MHC in representative samples from each of the three TG groups and the NTG control group is presented in Fig. 5A. Figure 5B shows the percent beta -MHC present in NTG and all TG groups calculated from densitometric analyses of the gels. There was a significant increase in the relative amount of beta -MHC expressed in ventricles from all TG animals compared with <10% beta -MHC in NTG control mice. We would expect from previous studies (38) that this increase in beta -MHC in hearts from TG animals would result in a slower cross-bridge cycle rate and thus a reduced tension cost in the TG hearts as we have observed (Fig. 4).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5.   Relative amount of beta -myosin heavy chain (beta -MHC) present in hearts from NTG and TG mice expressing enteric gamma -actin. A: electrophoretic separation of alpha - and beta -MHC in representative samples from mouse ventricle, as described in MATERIALS AND METHODS. B: densitometric analysis of %beta -MHC in NTG and TG mice. NTG, n = 13; TG +/+, n = 9; TG +/-, n = 3; TG -/-, n = 3. Data are means ± SE. *P <=  0.001 compared with NTG.

To determine whether the change in tension cost observed in TG mice was simply due to the increased expression of beta -MHC in these hearts, we induced changes in myosin isoform population in a group of NTG control mice by placing them on a diet that included 0.15% PTU for up to 8 wk. At different times of exposure to PTU, fiber bundles were isolated from these hearts and subjected to the experimental protocols previously described. Relative amounts of alpha - and beta -MHC were also quantified, as shown in Fig. 6.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6.   Induction of beta -MHC expression in hearts from NTG mice by administration of propylthiouracil (PTU). A: electrophoretic separation of alpha - and beta -MHC in representative ventricular samples from mice exposed to PTU in the diet for 1-8 wk, as described in MATERIALS AND METHODS. Lane 1, untreated; lane 2,+PTU at 1 wk; lane 3, +PTU at 2 wk; lane 4, +PTU at 4 wk; lane 5, +PTU at 8 wk. B: densitometric analysis of %beta -MHC in NTG mice treated with PTU for different time periods. The number of determinations was 2-3 for each group, performed in duplicate. Data are means ± SE.

PTU treatment was very effective in causing the expression of beta -MHC, with a 25-fold induction of this protein isoform after 8 wk of PTU treatment. These animals provide a control group to determine if expression of enteric gamma -actin in the heart specifically contributes to the decreased tension cost observed in the TG mice. It is possible that PTU treatment may also reduce expression of skeletal alpha -actin mRNA in the heart (43). However, we estimate that our NTG mouse hearts contain <10% skeletal alpha -actin mRNA from preliminary Northern blot analysis of cardiac and skeletal alpha -actin mRNA levels (unpublished results). Moreover, a decrease in expression of the skeletal alpha -actin isoform in the PTU-treated hearts would tend to blunt rather than amplify the differences between TG and PTU-treated NTG animals noted in Fig. 7.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7.   The relation between tension cost and %beta -MHC in hearts from NTG, NTG treated with PTU, and TG mice expressing enteric gamma -actin. , NTG and NTG treated with PTU. , TG expressing enteric gamma -actin. Data were analyzed by multiple linear regression. The slopes of both regression lines indicated a significant (P <=  0.001) relation between tension cost and beta -MHC but did not significantly differ from each other (P <=  0.8). The y-intercept was significantly different (P <=  0.0001).

Fiber bundles from all three groups of TG animals were compared with fiber bundles from control NTG and PTU-treated NTG mice. The slopes of both regression lines were different from zero, indicating that beta -MHC is a factor in determining tension cost. More importantly, the slopes did not differ significantly from each other, indicating that effect of beta -MHC on tension cost is the same in both groups of animals. However, the two regression lines had significantly (P<= 0.0001) different elevations indicating that the decreased tension cost observed in the TG myofilaments is not merely due to the increased presence of beta -MHC. We estimated from the difference in the y-intercepts (NTG + PTU-treated NTG = 7.8 ± 0.23; TG = 6.60 ± 0.46) that the contribution made by enteric gamma -actin is ~50% of the total decrease in tension cost (Fig. 4). Thus a substantial proportion of the increased economy of force production seen in myofilaments from TG animals is due to changing the actin isoform. Moreover, the EC50 (µM Ca2+) of the Ca2+ dependence of tension development in myofilaments from NTG and NTG + PTU-treated mice was similar when myofilaments containing <5% beta -MHC (EC50 = 0.90 ± 0.08, n = 6) and those containing >60% beta -MHC (EC50 = 0.90 ± 0.05, n = 8) were compared. Thus it is also unlikely that the decreased sensitivity of fiber bundles from TG mice (Fig. 1) is due to an increase in beta -MHC.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results provide new insights into the contribution of actin to the economy of force production in cardiac myofilaments. Previous studies (1, 27, 40) reported that a shift to beta -myosin expression is associated with increased economy of tension development and a slower cross-bridge cycle rate primarily because of a decrease in the off rate. The additional decrease in tension cost that we observed in the presence of smooth muscle enteric gamma -actin clearly indicates that the properties and state of actin contribute to the rate of cross-bridge cycling independent of the myosin isoform. Our finding of a slowing of cross-bridge cycling rates in hearts expressing enteric gamma -actin is consistent with previous measurements of cardiac function in situ that demonstrated slowing in the rate of contraction and relaxation (21).

Although little attention has focused on the role of actin isoforms in determining the parameters of the cross-bridge cycle, due in part to the difficulty in exchanging actin isoforms within an otherwise constant environment, there are some data that provide support for findings reported here. Drummond et al. (8) reported that a single mutation in the actin gene of Drosophila alters cross-bridge kinetics. Conversion of Gly368 to Glu significantly reduced tension redevelopment after a quick stretch in demembranated muscle fibers and also reduced maximal tension development. In addition, Miller et al. (28), using the in vitro motility assay, demonstrated differences in sliding velocities between thin filaments containing either WT yeast actin and yeast actin with altered negatively charged NH2-terminal residues. On the other hand, motility assay measurements of Harris and Warshaw (15) detected no differences in sliding velocities between thin filaments containing either smooth or cardiac actin isoforms.

Modulation of opening and closing of the cleft between the upper and lower domains of myosin potentially provides a mechanism by which different actin isoforms could have an impact on the cross-bridge cycle rate. Although several regions on myosin interact with actin (31, 35), the main regions of interaction are the lower half of the 50K domain and the positively charged loop 2 region. The sites on actin that interact with these domains of myosin comprise subdomains 1 and 2, regions that also contain all of the amino acid differences between cardiac alpha -actin and enteric gamma -actins. The binding of actin to myosin at the loop 2 site is thought to represent a weak binding state, in which there are rapid on-off interactions between actin and myosin (4). The significant differences in length and charge distribution in loop 2 between cardiac and smooth muscle myosins (32) could affect the formation of the weak binding state. Two recent reports (18, 45), using different experimental approaches, suggest that closure of the major cleft between the 50K upper and lower domains is associated with the transition from weak to strong cross bridges. The integration of movement of this cleft into the chemomechanical steps in the cross-bridge cycle is illustrated by Gordon et al. (14). Because the 50K lower portion of this cleft and loop 2 interface with actin, it is conceivable that the differences in amino acid composition between cardiac and enteric gamma -actin could affect the closure or opening of the 50K cleft and thus the transition from the weak to the strong binding state. This hypothesis is supported by evidence showing that the primary effect of mutating Arg403Gln in cardiac beta -MHC is a three- to fourfold decrease in actin-activated ATPase activity and a fivefold decrease in the velocity of actin movement on myosin (37). The Arg403 mutation, which is highly penetrant and associated with familial hypertrophic cardiomyopathy (42), is close to the region linking the upper and lower 50K domains (11, 33).

In addition to alterations at the actin-myosin interface, substitution of enteric gamma -actin for cardiac alpha -actin in cardiac myofilaments may also affect the interaction of tropomyosin (Tm) and troponin I (TnI) with the thin filament. The amino acid differences between cardiac alpha -actin and enteric gamma -actin lie within the actin-myosin interface but also overlap with the region of actin binding to Tm (3, 19) and TnI. Lehman et al. (24) reported that variations in actin isoforms were able to modulate the localization of Tm on actin filaments. In the case of TnI, Levine et al. (26) localized the binding of TnI to NH2-terminal residues 1-7 and 19-44 of actin. Ca2+ binding to troponin C (TnC) with subsequent release of TnI from actin would facilitate interaction of the negatively charged NH2-terminal of actin with loop 2 on myosin forming weak actin-myosin interactions. Ca2+ binding to TnC also results in movement of tropomyosin on actin (23) and exposure of the myosin-binding site. Whether movement of actin subdomains occurs with Ca2+ activation is not certain. On the basis of the X-ray interpretation and optical diffraction data, Squire and Morris (36) have reported that this possibility exists. Yet measurements using fluorescent resonance energy transfer between probes in subdomains 1 and 2 of actin did not reveal any distance changes associated with Ca2+ activation of skeletal muscle preparations (12). Therefore, our finding of a decrease in the Ca2+ sensitivity of myofilaments containing gamma -actin may be related more closely with a modulation of the position of Tm on the thin filament as well as a tighter interaction of cardiac TnI with actin. Alterations in Ca2+ sensitivity coupled with changes in cross-bridge kinetics are likely to account for the slow rate of contraction seen in the intact heart (21). We find no evidence in isolated fiber bundles for alterations in Ca2+ sensitivity of tension development with changes in beta -myosin content. Thus, as one would predict, substitution of enteric gamma -actin for cardiac alpha -actin in cardiac fibers alters both the cross-bridge interaction with actin and its regulation by Ca2+.

The association of single site mutations in actin with both dilated and hypertrophic cardiomyopathies in humans emphasizes the importance of actin structure to the maintenance of contractile function in the heart. The Glu361Gly mutation associated with dilated cardiomyopathy (30) occurs in subdomain 1 of actin and abuts the substitution of cardiac alpha -actin Gln360 by Pro in enteric gamma -actin. Other point mutations in actin associated with cardiomyopathies in humans occur in subdomain 3 of actin except for Glu99Lys, which is also located in subdomain 1 at the actin-myosin interface. In this study, we show that the four amino acid substitutions and one amino acid deletion in smooth muscle gamma -actin significantly affect activation and the economy of force production in isolated cardiac myofilaments. This slowing of the cross-bridge cycle by the presence of enteric gamma -actin in the heart suggests that actin has a more prominent role in defining the kinetics of the cross-bridge cycle in muscle than previously considered.


    ACKNOWLEDGEMENTS

The authors thank Vlasios Manaves for assistance in the electrophoretic separation of alpha - and beta -MHCs.


    FOOTNOTES

* A. F. Martin and R. M. Phillips contributed equally to this study.

This work was supported by National Heart, Lung, and Blood Institute Grants PO1 HL-62426-01 (Project 1 to R. J. Solaro and A. F. Martin and Project 4 to P. de Tombe), R37 HL-22231 (to R. J. Solaro), RO1 HL-57291 (to J. L. Lessard), and T32 HL-07692 (to R. M. Phillips). P. de Tombe was an Established Investigator of the American Heart Association during this study.

Address for reprint requests and other correspondence: A. F. Martin, Dept. of Physiology and Biophysics, M/C 901, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612 (E-mail: afmartin{at}uic.edu).

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.

April 25, 2002;10.1152/ajpheart.00890.2001

Received 12 October 2001; accepted in final form 22 April 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alpert, NR, and Mulieri LA. The partitioning of altered mechanics in hypertrophied heart muscle between the sarcoplasmic reticulum and the contractile apparatus by means of myothermal measurements. Basic Res Cardiol 72: 153-159, 1977[ISI][Medline].

2.   Barany, M. ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol Suppl 50: 197-216, 1967.

3.   Bing, W, Razzaq A, Sparrow J, and Marston S. Tropomyosin and troponin regulation of wild type and E93K mutant actin filaments from Drosophila flight muscle. Charge reversal on actin changes actin-tropomyosin from on to off state. J Biol Chem 273: 15016-15021, 1998[Abstract/Free Full Text].

4.   Bobkov, AA, Bobkova EA, Lin SH, and Reisler E. The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head. Proc Natl Acad Sci USA 93: 2285-2289, 1996[Abstract/Free Full Text].

5.   Brenner, B, and Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci USA 83: 3542-3546, 1986[Abstract/Free Full Text].

6.   De Tombe, PP, and ter Keurs HE. Force and velocity of sarcomere shortening in trabeculae from rat heart. Effects of temperature. Circ Res 66: 1239-1254, 1990[Abstract/Free Full Text].

7.   De Tombe, PP, and Stienen GJM Protein kinase A does not alter economy of force maintenance in skinned rat cardiac trabeculae. Circ Res 76: 734-741, 1995[Abstract/Free Full Text].

8.   Drummond, DR, Peckham M, Sparrow JC, and White DC. Alteration in crossbridge kinetics caused by mutations in actin. Nature 348: 440-442, 1990[Medline].

9.   Fabiato, A, and Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol 75: 463-505, 1979.

10.   Garner, I, Minty AJ, Alonso S, Barton PJ, and Buckingham ME. A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue. EMBO J 5: 2559-2567, 1986[ISI][Medline].

11.   Geeves, MA, and Holmes KC. Structural mechanism of muscle contraction. Annu Rev Biochem 68: 687-728, 1999[ISI][Medline].

12.   Gerson, JH, Kim E, Muhlrad A, and Reisler E. Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions. J Biol Chem 276: 18442-18449, 2001[Abstract/Free Full Text].

13.   Godt, RE, and Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol 80: 279-297, 1982[Abstract/Free Full Text].

14.   Gordon, AM, Regnier M, and Homsher E. Skeletal and cardiac muscle contractile activation: tropomyosin "rocks and rolls." News Physiol Sci 16: 49-55, 2001[Abstract/Free Full Text].

15.   Harris, DE, and Warshaw DM. Smooth and skeletal muscle actin are mechanically indistinguishable in the in vitro motility assay. Circ Res 72: 219-224, 1993[Abstract/Free Full Text].

16.   Hewett, TE, Grupp IL, Grupp G, and Robbins J. alpha -Skeletal actin is associated with increased contractility in the mouse heart. Circ Res 74: 740-746, 1994[Abstract/Free Full Text].

17.   Hoh, JFY, McGrath PA, and Hale PT. Electrophoretic analysis of multiple forms of rat cardiac myosin: effects of hypophysectomy and thyroxine replacement. J Mol Cell Cardiol 10: 1053-1076, 1977[ISI].

18.   Holmes, KC, Schroeder RR, Stegmann H, and Baehler M. The structure of the actin-myosin strong binding state (Abstract). Biophys J 80: 198a-199a, 2001.

19.   Korman, VL, Hatch V, Dixon KY, Craig R, Lehman W, and Tobacman LS. An actin subdomain 2 mutation that impairs thin filament regulation by troponin and tropomyosin. J Biol Chem 275: 22470-22478, 2000[Abstract/Free Full Text].

20.   Korman, VL, and Tobacman LS. Mutations in actin subdomain 3 that impair thin filament regulation by troponin and tropomyosin. J Biol Chem 274: 22191-22196, 1999[Abstract/Free Full Text].

21.   Kumar, A, Crawford K, Close L, Madison M, Lorenz J, Doetschman T, Pawlowski S, Duffy J, Neumann J, Robbins J, Boivin GP, O'Toole BA, and Lessard JL. Rescue of cardiac alpha -actin-deficient mice by enteric smooth muscle gamma -actin. Proc Natl Acad Sci USA 94: 4406-4411, 1997[Abstract/Free Full Text].

22.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

23.   Lehman, W, Craig R, and Vibert P. Ca2+-induced tropomyosin movement in limulus thin filaments revealed by three-dimensional reconstruction. Nature 368: 65-67, 1994[Medline].

24.   Lehman, W, Hatch V, Korman V, Rosol M, Thomas L, Maytum R, Geeves MA, Van Eyk JE, Tobacman LS, and Craig R. Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. J Mol Biol 302: 593-606, 2000[ISI][Medline].

25.   Lessard, JL. Two monoclonal antibodies to actin: one muscle selective and one generally reactive. Cell Motil Cytoskeleton 10: 349-362, 1988[ISI][Medline].

26.   Levine, BA, Moir AJ, and Perry SV. The interaction of troponin-I with the N-terminal region of actin. Eur J Biochem 172: 389-397, 1988[ISI][Medline].

27.   Loiselle, DS, Wendt IR, and Hoh JF. Energetic consequences of thyroid-modulated shifts in ventricular isomyosin distribution in the rat. J Muscle Res Cell Motil 3: 5-23, 1982[ISI][Medline].

28.   Miller, CJ, Wong WW, Bobkova E, Rubenstein PA, and Reisler E. Mutational analysis of the role of the N terminus of actin in actomyosin interactions. Comparison with other mutant actins and implications for the cross-bridge cycle. Biochemistry 35: 16557-16565, 1996[Medline].

29.   Olson, TM, Doan TP, Kishimoto NY, Whitby FG, Ackerman MJ, and Fananapazir L. Inherited and de novo mutations in the cardiac actin gene cause hypertrophic cardiomyopathy. J Mol Cell Cardiol 32: 1687-1694, 2000[ISI][Medline].

30.   Olson, TM, Michels VV, Thibodeau SN, Tai YS, and Keating MT. Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science 280: 750-752, 1998[Abstract/Free Full Text].

31.   Rayment, I, Holden HM, Whittaker M, Yohn CB, Lorenz M, Holmes KC, and Milligan RA. Structure of the actin-myosin complex and its implications for muscle contraction. Science 261: 58-65, 1993[Abstract/Free Full Text].

32.   Rovner, AS. A long, weakly charged actin-binding loop is required for phosphorylation-dependent regulation of smooth muscle myosin. J Biol Chem 273: 27939-27944, 1998[Abstract/Free Full Text].

33.   Ruppel, KM, and Spudich JA. Structure-function analysis of the motor domain of myosin. Annu Rev Cell Dev Biol 12: 543-573, 1996[ISI][Medline].

34.   Schurch, W, Skalli O, Seemayer TA, and Gabbiani G. Intermediate filament proteins and actin isoforms as markers for soft tissue tumor differentiation and origin. I. Smooth muscle tumors. Am J Pathol 128: 91-103, 1987[Abstract].

35.   Sheterline, P, Clayton J, and Sparrow JC. Actin. Protein Profile 2: 1, 1995[ISI][Medline].

36.   Squire, JM, and Morris EP. A new look at thin filament regulation in vertebrate skeletal muscle. FASEB J 12: 761-771, 1998[Abstract/Free Full Text].

37.   Sweeney, HL, Straceski AJ, Leinwand LA, Tikunov BA, and Faust L. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J Biol Chem 269: 1603-1605, 1994[Abstract/Free Full Text].

38.   Swynghedauw, B. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev 66: 710-771, 1986[Abstract/Free Full Text].

39.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract/Free Full Text].

40.   Van der Velden, J, Moorman AF, and Stienen GJ. Age-dependent changes in myosin composition correlate with enhanced economy of contraction in guinea-pig hearts. J Physiol 507: 497-510, 1998[Abstract/Free Full Text].

41.   Von Arx, P, Bantle S, Soldati T, and Perriard JC. Dominant negative effect of cytoplasmic actin isoproteins on cardiomyocyte cytoarchitecture and function. J Cell Biol 131: 1759-1773, 1995[Abstract/Free Full Text].

42.   Watkins, H, Rosenzweig A, Hwang DS, Levi T, McKenna W, Seidman CE, and Seidman JG. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N Engl J Med 326: 1108-1114, 1992[Abstract].

43.   Winegrad, S, Wisnewsky C, and Schwartz K. Effect of thyroid hormone on the accumulation of mRNA for skeletal and cardiac alpha -actin in hearts from normal and hypophysectomized rats. Proc Natl Acad Sci USA 87: 2456-2460, 1990[Abstract/Free Full Text].

44.   Wolska, BM, Keller RS, Evans CC, Palmiter KA, Phillips RM, Muthuchamy M, Oehlenschlager J, Wieczorek DF, de Tombe PP, and Solaro RJ. Correlation between myofilament response to Ca2+ and altered dynamics of contraction and relaxation in transgenic cardiac cells that express beta -tropomyosin. Circ Res 84: 745-751, 1999[Abstract/Free Full Text].

45.  Yengo CM, De La Cruz EM, Chrin LR, Gaffney DP II, and Berger CL. Actin-induced closure of the actin-binding cleft of smooth muscle myosin. J Biol Chem. In press.

Am J Physiol Heart Circ Physiol 283(2):H642-H649
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. C. G. Daniels, T. Naya, V. L. M. Rundell, and P. P. de Tombe
Development of contractile dysfunction in rat heart failure: hierarchy of cellular events
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R284 - R292.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
A. Thawornkaiwong, J. Pantharanontaga, and J. Wattanapermpool
Hypersensitivity of myofilament response to Ca2+ in association with maladaptation of estrogen-deficient heart under diabetes complication
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R844 - R851.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
S. Clement, M. Stouffs, E. Bettiol, S. Kampf, K.-H. Krause, C. Chaponnier, and M. Jaconi
Expression and function of {alpha}-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation
J. Cell Sci., January 15, 2007; 120(2): 229 - 238.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
J. E. Stelzer, D. P. Fitzsimons, and R. L. Moss
Ablation of Myosin-Binding Protein-C Accelerates Force Development in Mouse Myocardium
Biophys. J., June 1, 2006; 90(11): 4119 - 4127.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
V. L. M. Rundell, V. Manaves, A. F. Martin, and P. P. de Tombe
Impact of {beta}-myosin heavy chain isoform expression on cross-bridge cycling kinetics
Am J Physiol Heart Circ Physiol, February 1, 2005; 288(2): H896 - H903.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
V. L. M. Rundell, D. L. Geenen, P. M. Buttrick, and P. P. de Tombe
Depressed cardiac tension cost in experimental diabetes is due to altered myosin heavy chain isoform expression
Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H408 - H413.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. B. Marston and C. S. Redwood
Modulation of Thin Filament Activation by Breakdown or Isoform Switching of Thin Filament Proteins: Physiological and Pathological Implications
Circ. Res., December 12, 2003; 93(12): 1170 - 1178.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/2/H642    most recent
00890.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map