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1Department of Biochemistry and Molecular Genetics and 2Department of Physiology and Biophysics, University of Illinois College of Medicine, Chicago, Illinois 60612
Submitted 8 September 2003 ; accepted in final form 3 December 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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actin nucleotide; nucleotide exchange
Actin is a unique protein that exists in two forms, globular (G) monomer and filamentous (F) polymer. G-actin contains bound ATP, which, on polymerization, is transformed into F-actin-bound-ADP and Pi (31). It is generally accepted that actin polymerization is the basic mechanism for motility in nonmuscle cells (10, 27). On the other hand, no convincing evidence has been obtained for actin polymerization in skeletal muscle (24).
Mehta and Gunst (25) and Jones et al. (17) were the first to report the presence of G-actin in smooth muscle with the methods of DNase inhibition and phalloidin staining, respectively. We examined the G-actin/F-actin distribution in smooth muscle by labeling the muscle with 32P and separating the free nucleotides from the actin-bound nucleotides by an alcohol-water extraction technique (3). Briefly, 32P labeling results in the synthesis of [
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-32P]ATP in the cytosol, which exchanges with G-actin bound ATP. Polymerization of G-actin into F-actin results in F-actin-bound [-32P]ADP. Hence, 32P labeling of actin allows for the determination of steady-state G-actin monomer to F-actin polymer distribution. Using this method we showed exchange of the actin-bound nucleotide with cytoplasmic ATP in smooth muscle (3), thus demonstrating the presence of G-actin in smooth muscle. Subsequently, several laboratories reported actin polymerization in smooth muscle related to contractile activity: pressure-induced actin polymerization in cerebral arteries was suggested as a mechanism underlying myogenic behavior (9). It was also suggested that profilin plays a role in the regulation of actin polymerization in response to contractile stimulation of carotid arteries (33). Inhibitors of actin polymerization attenuated force in pressurized mesenteric arteries (30) or inhibited contractile differentiation of rat portal veins in organ culture (35). Thus these results suggest that polymerization of G-actin monomer into filamentous F-actin may play an important role in contractile activation in smooth muscle independent of activation of myosin ATPase activity in response to myosin light chain phosphorylation.
In contrast to the extensive work on actin polymerization in smooth muscle, virtually no information exists about this reaction in cardiac muscle. Nevertheless, it is generally assumed that the actin filament in striated muscle is a relatively stable cellular structure that does not undergo dynamic alterations within the time frame of one or several cardiac cycles. In the experiments reported here, we used our recently developed technique in smooth muscle (3) to determine the turnover rate of G-actin into F-actin in the heart. We used a perfused rat heart preparation to answer the following questions: First, what is the distribution between G-actin monomer and F-actin polymer in the heart? Second, what is the rate of G-actin polymerization to F-actin in the heart? Finally, does the contractile state of the heart affect this distribution or turnover rate?
We found that a significant amount of the actin-bound nucleotide was in the ATP form, suggesting the presence of a sizable G-actin momomer concentration in the heart. Furthermore, the transformation of G-actin into F-actin was too fast to be measured with our technique at 37°C and could only be assessed at 25°C. Finally, we found a reduction in G-actin content in contracting hearts vs. diastolic-arrested hearts. Our results suggest that a large fraction of actin in the heart is in the form of G-actin monomer and the rate of G-actin polymerization is regulated in conjunction with contractile state. Preliminary results of this work were published in abstract form (4).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The spontaneously beating heart was equilibrated with PSS under 95% O2-5% CO2, at a temperature of 25°Cor37°C until constant flow rate and beating rate were obtained (10–15 min). The normal PSS was then exchanged for PSS containing 1.0 mCi carrier-free [32P]orthophosphate, and the heart was perfused with aid of a Control Company model 3386 pump (Friendswood, TX) for 5 min. Subsequently, excess 32P from the extracellular space of the heart was washed out with normal PSS for 3.5 min. The same type of 32P perfusion was performed with hearts paced with a World Precision Instruments A320 stimulator (Sarasota, FL) or with the heart exposed to positive or negative inotropic agents. Under these conditions, the electrical stimulation or chemical treatment was continued during the wash period. Two hearts were used for each experiment, which was completed within 1 h (stock 32P solution was divided into 2 parts; each part was used for the perfusion of 1 heart). At the end of the experiment, the ventricle was isolated on an ice-cooled watch glass and cut in the middle. On a random basis, one half ventricle was placed into a blender and kept in ice and the other half ventricle was dropped into 100 ml of 75% methanol and kept in ice.
For perchloric acid (PCA) extraction, the ventricles were blended with 1.5% PCA at 0°C for 1 min. The suspension was centrifuged briefly, and the supernatant was poured into a small volume of 10 N KOH solution to neutralize PCA. The perchlorate precipitate was centrifuged off, and the specific activity (SA) of phosphocreatine (PCr) was determined from an aliquot of the supernatant (20); the remainder of the supernatant was concentrated by SpeedVac and used to determine the SA of ATP and ADP by Dowex-1 chromatography.
For the 75% methanol extraction (100 ml), the ventricles were transferred to a –15°C freezer and extracted for 2 days. The ventricles were reextracted twice, each with 100 ml of fresh 75% methanol at –15°C for 2 h. All of the free nucleotides were removed from the hearts by this procedure because reextraction of the residue did not release any material that absorbed at 260 nm. It was demonstrated previously that repeated alcoholic extraction of smooth muscle removes all the free nucleotides but not the actin-bound nucleotides (3). The methanol-treated ventricles were extracted with 1.5% PCA at 0°C for 1 h. After centrifugation, the supernatant was neutralized and centrifuged and the supernatant was concentrated for Dowex-1 chromatography. In most experiments, 75% methanol extraction at –15°C was used to analyze the actin-bound nucleotides; similar results were obtained when the extraction was carried out with 65% or 70% methanol at –15°C or with 65–75% methanol at 0°C.
Dowex-1 chromatography. The procedure was described previously (3). Figure 1 illustrates the elution pattern of compounds that absorbed at 260 nm and their radioactivity in the neutralized PCA extract. NAD was eluted in the early part of the chromatogram, and it was radioactive. Pi, ADP, and ATP were also radioactive. The elution pattern of the 32P-labeled actin-bound nucleotides is shown in Figs. 3 and 4.
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Determination of exchange of actin-bound nucleotides in perfused heart. The percent exchange of the actin-bound ATP and ADP was calculated as
 | (1) |
 | (2) |
-P is the middle phosphate of ATP and
 | (3) |
Preparation of rat heart actin. Fresh hearts were washed with cold PSS to remove blood, and the ventricles were minced with scissors. The mince was extracted with 3 vols of 0.35 M KCl and 0.15 M KPi, pH 6.5, at 0°C for 20 min; after centrifugation the residue was washed with 5 vols of 0.4% NaHCO3 at 0°C for 10 min, followed by a 50-min wash with 10 vols of distilled water. Five treatments were performed with cold acetone, 5, 3, and 1 vol, each for 10 min. The dried powder was extracted with 30 vols of 0.5 mM ATP at 0°C for 1 h. Actin in the supernatant was polymerized by 0.1 M NaCl and 2 mM MgCl2 at 37°C for 1 h and then left at 0°C overnight. NaCl was added to the F-actin solution to 0.6 M, and it was stirred at 0°C for 30 min and then centrifuged at 30,000 rpm at 5°C for 3 h. The pellet was washed with distilled water to remove the salts and dissolved by homogenization in 0.5 mM ATP to yield a nonviscous G-actin (4–5 mg/ml).
The dried powder previously extracted with 0.5 mM ATP was further extracted with 30 vols of a solution containing 0.25 M KI, 0.02 M Tris·HCl, pH 7.4, and 1 mM ATP (modified from Ref. 2) at 0°C for 1 h. The supernatant was dialyzed against a solution of 0.1 M NaCl and 2 mM MgCl2, with frequent changes, at 4°C for 20 h. The workup of the F-actin solution was the same as described in the previous paragraph. On addition of salts to 0.1 M NaCl and 2 mM MgCl2, this actin solution became very viscous. This actin migrated as a single band on acrylamide gels (see Figs. 8 and 9).
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Exchange of actin-bound nucleotides in vitro. [,-32P]ATP and [-32P]ADP were prepared by perfusing rat hearts with 2 mCi 32Pi at 37°C for 30 min and isolating the nucleotides by Dowex-1 chromatography. The SA of [,-32P]ATP was in the range of 8 x 107 cpm/µmol and that of [-32P]ADP was 4 x 107 cpm/µmol. The labeled nucleotides were diluted with the corresponding unlabeled nucleotides to have a SA of 
100,000 cpm/µmol ATP and 50,000 cpm/µmol ADP in the exchange reaction mixture [conditions: G- or F-actin, 2.5–3 mg/ml; molar ratio of 32P-labeled free nucleotide to actin-bound nucleotide, 15–20; 18 mM NaCl, 2 mM Tris·HCl, 2 mM MgCl2 (in case of F-actin), pH 7.1; 25°C].
Before the experiment started, free nucleotides were removed from the actin solutions by treatment with Dowex-1 X8, 200–400 mesh, 25 mg/ml (modified from Ref. 5) and the reaction was initiated by the addition of actin. Three-milliliter aliquots were removed from the reaction mixture at 30-s and 2-, 4-, 8-, and 16-min intervals and quickly mixed with 80 mg of Dowex-1 to stop the exchange reaction. Three-milliliter aliquots containing no actin were also treated with Dowex-1 to obtain blank values. The resin was removed by brief centrifugation at 400 g, and the upper two-thirds of the supernatant was taken for analyses. PCA was added to the supernatant to release the actin-bound nucleotides, and after centrifugation absorbance at 260 and 280 nm and radioactivity were measured in the supernatant. The actin-containing precipitate was dissolved in 1 N NaOH, and the protein content was determined. From these data the number of moles of bound nucleotide per 42,000 g of actin was calculated; the values ranged from 0.74 to 1.29.
In calculating the SA of the actin-bound nucleotide, the small blank absorbance and radioactivity values were subtracted from the values measured for the bound nucleotides. Next, the SA of the actin-bound nucleotide was compared with the SA of [,-32P]ATP or [-32P]ADP in the medium; this term is called percent exchange
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 | (5) |
Preparation of myofibrils from 32P-labeled rat hearts. Ventricles from 32P-perfused hearts were chopped with scissors. A part of the mince was treated with PCA for determination of the SA of PCr and ATP. The rest of the mince was blended at 0°C with a solution containing 0.01 M iodoacetamide, 0.04 M NaCl, and 0.01 M Tris·HCl, pH 7.4, for 15 s (iodoacetamide in this solution inhibits the enzymic hydrolysis of ATP). The suspension was centrifuged at 600 g for 25 min at 4°C, and the residue was blended with 0.04 M NaCl and 0.01 M Tris·HCl for 1 s and then centrifuged as before. This step was repeated. The final pellet was suspended in the NaCl-Tris·HCl solution and clarified at 100 g to remove connective tissue. The myofibrils in the supernatant were analyzed for bound nucleotide content, composition of the nucleotides, and their radioactivity.
Preparation of mitochondria from 32P-labeled rat hearts. Ventricles from 32P-perfused hearts were blended at 0°C with a solution containing 0.1 M NaCl and 0.05 M Tris·HCl, pH 7.4, in two bursts, each lasting 30 s. The homogenate was centrifuged at 600 g, and the mitochondria were isolated from the supernatant by centrifugation at 10,000 g. The pellet was repeatedly homogenized at 0°C in NaCl-Tris, with the aid of a glass Potter-Elvehjem homogenizer, and centrifuged at 10,000 g. Mitochondria were also prepared from 32P-labeled rat hearts by sucrose-sorbitol homogenization (13). No difference was found in the 32P content of mitochondria prepared by the two methods.
Determination of free nucleotide content of rat hearts. With repeated 75% methanol extraction, 5.82 ± 0.65 µmol of adenine nucleotides were obtained per gram of heart (n = 14). This value included the NAD and NADP content of the heart, 0.77 µmol/g (22). The ATP content of the methanol extract was 4.0 µmol/g. With 31P-NMR, 5.4 µmol of ATP in 73% intracellular water of the rat heart (3.94 µmol ATP/g heart) was reported (12).
Determination of bound nucleotide content of rat hearts. Weighed pieces of ventricles (230–270 mg) were extracted with 30 ml of 0.04 M NaCl and 0.01 M Tris·HCl, pH 7.4, with stirring at 4°C for 16–18 h. After centrifugation at 600 g, the residue was extracted twice with the NaCl-Tris solution (no free nucleotide remained in the last supernatant). The final residue was treated with PCA to liberate the actin-bound nucleotides. A value of 1.03 ± 0.12 µmol nucleotide/g wet heart weight was found (n = 10). With 75% methanol extraction and subsequent PCA treatment, 1.06 ± 0.08 µmol bound nucleotide/g heart was found (n = 21). As shown under Determination of free nucleotide content of rat hearts, rat hearts contain 4 µmol free nucleotides/g, thus the actin-bound nucleotides comprise 20% of the total nucleotides.
Quantifying the actin content of rat hearts by gel electrophoresis gave 38.8 mg actin/g heart. With a molecular mass of 42 kDa for heart actin (7), 0.92 µmol of actin monomer was found per gram of heart. This compares well with the 1.03 and 1.06 µmol actin/g heart measured by the bound nucleotide method.
Determination of myosin content of rat hearts. This procedure is based on the observations (6, 32) that a muscle washed free of ATP and subsequently extracted with 0.6 M KI solution yields actomyosin containing monomeric actin. On dilution to 0.024 M KI, the myosin component of the actomyosin precipitates while the actin component of the actomyosin remains in the supernatant. We found 77.3 ± 12.1 mg myosin/g wet heart weight (n = 6). Because the heart contains 42 mg actin/g, there is a 2.6-fold molar excess of actin monomer over the myosin heavy chain (molecular mass 200 kDa) in the rat heart.
Determination of rat heart wet weight. Weighed ventricles were extracted with 100 vols of 0.1 N NaOH solution at 25°C for 24 h (21). After centrifugation to remove connective tissue, 184.9 ± 6.5 mg protein/g heart remained in the supernatant (n = 10). This value is somewhat higher than the 160 mg protein reported (18). In our experiments, the heart residue left over after 75% methanol or 1.5% PCA extraction was extracted with 0.1 N NaOH and the amount of protein solubilized was converted into grams of wet heart weight with the factor of 184.9.
Miscellaneous. The band densities of proteins on gels were determined by digital analysis with a Kodak Digital Science DC 120 Zoom digital camera and Kodak Digital Science 1D 2.0.2 software (Eastman Kodak). The protein content of the heart preparations was determined by the modified biuret method (1) after dissolution in 0.1 N NaOH. Radioactivity was measured by liquid scintillation counting. 32Pi was obtained from ICN. Data are expressed as means ± SE.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,-32P]ATP was investigated (Fig. 2). The percent exchange (Eq. 4) was 92–95% for G-actin and virtually zero for F-actin. Under the conditions of Fig. 2, 20-fold molar excess of [,-32P]ATP was used over the molar concentration of the actin-bound nucleotide; thus the maximal theoretical percent exchange was 95%. The exchange was immediate; that is, at the earliest experimental time (30 s), maximal exchange occurred. The same exchange pattern was found at 37°C as at 25°C; i.e., there was no exchange for F-actin, whereas there was full exchange for G-actin.
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When [,-32P]ATP was substituted with [-32P]ADP, no exchange was found with F-actin-ADP at either 25°C or 37°C (calculated with Eq. 5) but considerable exchange was found with G-actin-ATP. From these data one can conclude that the F-actin-ADP is not exchangeable with either ATP or ADP in the medium. These data also show that the exchangeability of heart actin is the same as that of skeletal muscle actin (23, 24).
Distribution of actin-bound nucleotides in perfused hearts. Two bound nucleotides, ADP and ATP, and three radioactive peaks, Pi, ADP, and ATP, were found by Dowex-1 chromatography (Figs. 3 and 4). The absorbance of the actin-bound ATP was higher than that of the actin-bound ADP. A comparison of Fig. 3 with Fig. 4 revealed that the ratio of the actin-bound ATP to the actin-bound ADP depended on the functional state of the heart. Thus, from the areas of the ATP and ADP peaks, a contribution of 54% for ATP and 46% for ADP was determined in the heart stimulated with isoproterenol (Fig. 3), whereas in the heart stopped in diastole with excess Mg2+ the contribution was as much as 77% for ATP and only 23% for ADP (Fig. 4).
In Fig. 5 the percentage distributions of ATP and ADP were compared between hearts stopped reversibly in diastole by low Ca2+, high Mg2+, or high K+ and hearts stimulated by isoproterenol, high Ca2+, or electrical pacing. In the resting heart the distribution was ATP 75 ± 6% and ADP 25 ± 4%, whereas in the stimulated heart the distribution was ATP 53 ± 7% and ADP 47 ± 8%.
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Exchange of actin-bound nucleotides in 32P-perfused hearts. The maximal radioactivity in the ATP peaks matched the maximal absorbance in both stimulated and resting hearts (Figs. 3 and 4), indicating a high level of 32P incorporation into the actin-bound ATP. On the other hand, the radioactivities of the ADP peaks remained below the maxima of the corresponding absorbance peaks, suggesting that the 32P incorporation into the actin-bound ADP was not complete. This reveals a difference in the labeling pattern between bound ATP and bound ADP.
To quantify the incorporation of 32Pi into the actin-bound nucleotides, we measured the SA of their 32P label and compared it with the SA of the 32P label in the cytoplasmic nucleotides (Eqs. 1 and 2). The exchange was so rapid at 37°C that its kinetics could not be measured. Therefore, we lowered the perfusion temperature from 37°C to 25°C.
In spontaneously beating hearts, the complete exchange of actin-bound ATP was immediate whereas the full exchange of the bound ADP took over 20 min (Fig. 6). The exchange of the bound ATP occurred at the lowest stimulation frequency, whereas the full exchange of the bound ADP required a stimulation frequency of 200 min–1 (Fig. 7). From the profiles of Figs. 6 and 7 one can conclude that the exchange of the actin ATP is a faster reaction than the exchange of the actin ADP.
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Mitochondria. The heart is rich in mitochondria (19). We have found on average 13 mg mitochondrial protein/g rat heart, containing a total of 0.04 µmol of bound ATP and bound ADP. When isolated from 32P-labeled heart, the mitochondrial ATP and ADP were fully exchanged. Because the actin-bound nucleotide amounts to 1 µmol/g heart, the small quantity of mitochondrial ATP and ADP has only a negligible effect on the conclusions drawn in the present work.
Myofibrils. Myofibrils from hearts perfused with 32Pi contained 0.6–0.75 µmol bound 32P-ADP/100 mg protein. Full exchange of the myofibril-bound ADP was reached in spontaneously beating hearts in 10 min.
Analysis of PCA extract. 31P-NMR studies on hearts of live animals and on perfused hearts indicated that "Pi is virtually undetectable" (Ref. 14 and references therein) because of the rapid incorporation of Pi into ADP formed during cellular metabolism. Therefore, the ADP found in the PCA extract of a well-oxygenated, perfused rat heart is essentially the actin-bound ADP; this was shown to be the case in smooth muscle (3). The current study with rat hearts also confirmed that the PCA extract contained actin-bound ADP. For instance, the percent exchange of the actin-ADP in hearts stimulated at a frequency of 300 min–1 was 101 ± 5% when analyzed by the methanol-water extraction method and 95 ± 7% when measured by the PCA extraction method.
Evidence for existence of G-actin in rat heart muscle. When ventricles were homogenized in 0.15 M NaCl and 0.01 M Tris·HCl, pH 7.4, and centrifuged at 600 g, the supernatant contained on average 52 mg cytosolic proteins/g heart. Gel electrophoresis of the supernatant showed a strong band in the 42-kDa region (Fig. 8), with mobility identical to that of rat heart actin. Immunoblotting of the supernatant gel showed that the monoclonal antibody specific to sarcomeric actin stained a single band with mobility identical to that of rat heart actin (Fig. 8).
The staining intensity of the 42-kDa band was determined in four different experiments, and it was found to be 24.4 ± 3.0% of the total staining intensity of all the bands. This means that 12.7 mg of actin was present in the cytosol per gram of heart ventricle, or 30% of the total actin (42 mg).
Viscometric studies were used to establish the form of actin, G or F, in the supernatant. The viscosity was expressed as a logarithm of the relative viscosity, which is proportional to the protein concentration in actomyosin systems (28). The viscosity of the actin in the supernatant was about one-eighth of the viscosity of the pure rat cardiac F-actin, indicating that the actin in the supernatant was in the G form. The fact that actin in the supernatant in 0.15 M NaCl remained in the G form suggests that there is an inhibitor present in rat heart cytosol that prevents the polymerization of actin.
On the basis of nucleotide analysis (Figs. 3, 4, 5), it was postulated that the 75% methanol-extracted residue of the rat heart contains G-actin. Gel electrophoresis provided direct evidence for the existence of G-actin in this residue (Fig. 9). In both the first and second distilled water extracts of the methanolic residue, the strongest-stained band migrated with a mobility identical to that of pure rat heart actin. The staining intensity of the actin band in the water extracts was 55% of the total band intensities. Along with the proteins, nucleotides were also extracted from the methanolic residue, which were identified predominantly as ATP on the Dowex-1 column. Furthermore, the water extracts possessed low viscosity. All these characteristics suggest that the water extracts contained G-actin.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following observations support cardiac actin dynamics. 1) The actin-bound nucleotide in the rat heart exists in two forms, ATP and ADP. The ratio of bound ATP to bound ADP depended on the functional state of the heart; it was higher in hearts stopped in diastole than in stimulated hearts (Fig. 5). It is conventional to associate ATP-actin with G-actin and ADP-actin with F-actin; accordingly, the nucleotide data indicate that rat heart actin occurs both in the G and F forms. 2) Both actin-bound ATP and ADP were labeled in hearts perfused with PSS containing 32Pi. The labeling of the bound nucleotides was so fast that with our technique it could not be followed at 37°C, only at 25°C. The labeling of the bound ATP was immediate, whereas the labeling of the bound ADP lagged behind (Figs. 6 and 7). To explain these data, one must recall that ATP bound to rat heart G-actin readily exchanges with free ATP in the medium but ADP bound to rat heart F-actin does not exchange with free nucleotides (Fig. 2). The labeling of actin-bound ADP in the perfused heart can be described by the following reactions
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 | (7) |
,-32P]ATP is hydrolyzed to F-actin-[-32P]ADP and [32P]phosphate (Eq. 7). That is, the F-actin-ADP does not exchange directly, but the [32P]ADP label is introduced into F-actin through the polymerization of G-actin-[,-32P]ATP. Evidence was presented for the existence of G-actin protein per se in the cytoplasm of the heart by immunoblotting and gel electrophoresis (Fig. 8) and viscometry. As a matter of fact, as much as 30% of the total rat heart actin was found to be in the G form in the cytoplasm. As described under MATERIALS AND METHODS, there is a 2.6-fold molar excess of actin monomer over the myosin heavy chain in the rat heart and, therefore, a significant part of the actin could be in the G form. It should be noted that our value of the 30% actin content of heart cytoplasm is considerably higher than the 13% value reported by Seidel et al. (29). This difference is likely due to differences in the methods used: we separated the proteins by gel electrophoresis and scanned the intensity of the protein bands, whereas Seidel et al. measured DNase activity in the cytoplasm and compared it with that of pure G-actin. We believe that the analytical gel electrophoresis is more accurate than the comparative enzymic method.
Based on the treadmilling process (26, 34), Fig. 10 depicts a scheme of how these reactions may take place in the perfused heart. The double-labeled 32,32ATP in the cytoplasm exchanges with the bound ATP of free G-actin. Individual G-actin-32,32ATP molecules are attaching to the barbed end of the F-actin double helix in the thin filament while hydrolyzing the bound double-labeled ATP to single-labeled bound ADP. (The transformation of bound ATP to bound ADP is not shown in Fig. 10. Furthermore, Fig. 10 shows the attachment of the G-32,32ATP only to one of the twin strings of F-actin; in reality, the attachment to both strings takes place on a random basis.) To keep the filament length constant (under physiological conditions), a nonlabeled ADP-actin monomer must leave the actin string at the pointed end. The G-actin-ADP in the cytoplasm exchanges its nucleotide to be transformed to G-actin-32,32ATP; this ends the cycle.
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There are reports in the literature that treadmilling is not involved in nucleotide exchange of F-actin (8, 11). These experiments were carried out in solution and lasted for several hours, unlike the nucleotide exchange in the beating heart, which takes place in the thin filament compartment of the heart and is completed in minutes. Furthermore, the bound nucleotide of rat heart F-actin did not exchange with free nucleotides (Fig. 2). In analyses of polymerization dynamics (15, 16), diffusion exchange was preferred over treadmilling. We believe, however, that a random diffusion process would allow for less control over the obligatory sequence of actin polymerization in the structure of thin filaments than that provided by treadmilling.
The cycling rate of the G-actin-ATP to F-actin-ADP transformation was very fast at a physiological temperature (37°C). Moreover, the dynamic rate of actin monomer incorporation into the thin filament appears to be regulated in conjunction with the contractile state. Site-directed polymerization of actin in cardiac thin filaments, in response to stimulation, generates a movement (treadmilling) of the actin helix from the Z line toward the center of the sarcomere. The ongoing replacement of actin monomers may facilitate cross bridge formation by increasing the chance for a myosin head to be in optimal juxtaposition to actin and thereby enhance myofilament force development. One may speculate, therefore, that enhanced actin treadmilling would lead to enhanced contractility. It should be noted that the estimated rate of actin incorporation into the thin filament at 25°C amounted to 1 cycle per 3 s. This implies that this mechanism operates over the time course of several beats. However, we were not able to determine the rate at 37°C. We cannot, therefore, exclude a role for this process during the cardiac cycle at physiological temperatures.
In summary, we found a significant amount of G-actin monomer in the heart that is rapidly transformed into F-actin in the cardiac sarcomere. Furthermore, the rate of actin polymerization increased in conjunction with the contractile state. Our results suggest that the actin monomer-ATP to actin polymer-ADP transformation is of physiological significance in the heart.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Heart, Lung, and Blood Institute P01-HL-62426 Project 4 (to P. P. de Tombe).
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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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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Absorbance; 
, counts per milliliter.
