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Age-Related Decline in Actomyosin Function

¹ Department of Biochemistry, Molecular Biology, and Biophysics
² Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis.

Address correspondence to Ewa Prochniewicz, Department of Biochemistry, University of Minnesota, Jackson Hall 6-155, 321 Church Street, Minneapolis, MN 55455. E-mail: ewa{at}ddt.biochem.umn.edu

	Abstract

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To understand the molecular basis of the functional decline in aging muscle, we examined the functional (actomyosin ATPase) and chemical (cysteine content) changes in actin and myosin purified from the muscles of young (4- to 12-month-old) and old (27- to 35-month-old) Fisher 344 rats. Using the soluble, catalytically active myosin fragment, heavy meromyosin (HMM), we determined the maximum rate (V_max) and actin concentration at half V_max (K_m) of the actomyosin ATPase, using four combinations of actin and HMM from old and young rats. V_max and K_m were significantly lower when both actin and HMM were obtained from old rats than when both proteins were obtained from young rats. The number of reactive cysteines in HMM significantly decreased with age, but no change was detected in the number of reactive cysteines in actin. We conclude that aging results in chemical changes in myosin (probably oxidation of cysteines) that have inhibitory effects on the actin-activated myosin ATPase.

Among the hypotheses that have been developed to explain age-related functional changes in proteins, the one most relevant for this work involves oxidative modification of proteins by reactive oxygen species, resulting in inhibition of biological functions (7). We are particularly interested in the oxidative state of the cysteines in myosin and actin, as cysteine thiols are an important oxidation target associated with aging (8,9). Skeletal muscle myosin contains 40 cysteines (10), including two highly reactive cysteines, Cys 707 and Cys 697. Modification of these two cysteines has been shown to have dramatic effects on the functional properties of myosin, particularly in interactions with actin; spectroscopic probes attached to these sites are sensitive to ATPase activity and to actin binding (11–16). Actin contains five cysteines (17), including the highly reactive Cys 374, which is located in the region involved in the interaction with myosin. Modification of Cys 374 has been shown to affect functional interaction with myosin (18), and spectroscopic probes attached to this site are sensitive to functional interactions with myosin (19,20).

Investigations elucidating the mechanisms of the age-related degeneration of muscle have typically focused on myosin (21–24), but there is abundant evidence that modifications of actin affect actomyosin ATPase and in vitro motility (20,25–27). Therefore, to determine independently the roles of actin and myosin in age-related changes in muscle contractility, it is necessary to perform mechanical measurements on muscle from young and old animals, then purify actin and myosin from these same animals and determine separately their functional properties in the actomyosin ATPase reaction. Until the present study, this set of experiments has not been done.

In the present study, we tested the hypothesis that the age-related deterioration of muscle contractility is due to changes in the interaction between actin and myosin during the actomyosin ATPase cycle. It is difficult to study quantitatively these interactions using intact myosin, because it aggregates at low ionic strength where the interaction with actin is maximized. Therefore, quantitative measurements of the actin–myosin interaction are usually performed using soluble, catalytically active myosin fragments: two-headed heavy meromyosin (HMM) and one-headed subfragment 1 (S1). Kinetic studies have established that the mechanism of interaction between purified actin and soluble myosin heads in solution is essentially the same as that between actin and intact myosin in myofibrils (28). In the present study we purified actin and myosin from the muscles of young and old rats, prepared HMM, and determined two key parameters of the actomyosin ATPase, V_max (the maximum rate) and K_m (actin concentration at half V_max). We complemented these functional studies by measuring age-related changes in the content of reactive cysteine residues in purified actin and myosin. This approach represents a new step in exploring the mechanisms of age-related decline of actomyosin function.

	METHODS

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Animals
Fisher 344 male and female rats aged 4–12 months (young) and 27–35 months (old) were obtained from the aging colony maintained by the Minneapolis Veterans Administration. The 50% survival rate for the Fisher 344 rat strain is about 24 months. All animals were housed in pathogen-free conditions and received food and water ad libitum. The animal care protocol was approved by the University of Minnesota Institutional Animal Care and Use Committee. Animals were deeply anesthetized and killed, and the semimembranosus, semitendinosus, adductor magnus, and quadriceps muscles were dissected.

Single Fiber Contractile Measurements
Individual fiber segments (2 mm long) from the permeabilized bundles were isolated and studied at 15°C as described in detail previously (2). Fiber segments were mounted in relaxing buffer (7.0 mM EGTA, 5.4 mM MgCl₂, 20 mM imidazole [pH 7.0], 14.5 mM creatine phosphate, 4.7 mM ATP, CaCl₂ to achieve pCa of 9, and enough KCl to achieve ionic strength of 180 mM). Sarcomere length was set to 2.5 µm, the diameter was measured at three places along the length of the fiber, and then maximal isometric force was determined in activating solution. Unloaded maximal shortening velocity, V_o, was determined by the slack test, in which fibers were activated by exposure to Ca²⁺ (calcium added to the relaxing buffer to bring pCa to 4.5) and then at peak isometric force rapidly shortened (slacked) by 10%–20% of fiber length such that force dropped to zero. The time between zero force and force redevelopment was measured. This procedure was performed 5 times at different slack distances. The slack distances were then regressed against the corresponding times of force redevelopment, and the slope of that line (in millimeters per second) was divided by fiber length (millimeters per fl) to obtain V_o (fiber length in millimeters per second).

Purification of Myosin and Actin
Each set of experiments consisted of determining actin-activated HMM ATPase under physiological conditions, myosin and HMM ATPase under "high-salt" conditions, and cysteine content in actin and HMM. For each set, two rats (one old and one young) were killed. Muscles were quickly dissected and immediately placed in ice-cold solution containing 0.3 M sucrose, 0.1 mM EDTA, and 10 mM imidazole (pH 7.0); they were subsequently homogenized on ice in rigor buffer containing 0.1 M KCl, 20 mM imidazole (pH 7.0), 0.5 mM EDTA, and the following protease inhibitors: 20 µM PMSF (phenylmethanesulfonyl fluoride), and 10 µg/ml each of aprotinin, leupeptin, antipain, pepstatin, chymostatin, TLCK (N-p-Tosyl-L-lysine-chloromethyl ketone), TPCK (N-p-Tosyl-L-phenylalanine chloromethyl ketone), and BAAE (N-Benzoyl-L-arginine ethyl). The homogenate was centrifuged for 5 minutes at 3000 g, supernatant was discarded, and the pellet was extracted for 10 minutes on ice with Guba-Straub solution (0.3 M KCl, 0.1 M KH₂PO₄, and 50 mM K₂HPO₄, pH 6.4). The extract was centrifuged for 5 minutes at 3000 g. The supernatant was used to prepare myosin, and the pelleted muscle residue was used to prepare acetone powder, following standard methods of preparation of myosin and actin from rabbit skeletal muscle (29,30). Myosin and actin from rabbit skeletal muscle were used in control experiments.

HMM, the soluble two-headed fragment of myosin, was prepared by -chymotryptic digestion of rat muscle myosin, following methods described for rabbit skeletal muscle myosin (31). The digest was dialyzed against 10 mM Tris (pH 7.5) and centrifuged for 45 minutes at 300,000 g to remove the myosin rods and undigested myosin. The resulting HMM was used without further purification. No difference in the digestion patterns of young and old myosin was observed on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis.

Actin was extracted from rat muscle acetone powder with G-buffer (5 mM Tris (pH 7.5), 0.2 mM CaCl₂, 0.2 mM ATP), and was purified by the method used for Drosophila (32). The extract was first polymerized with 0.1 M KCl for 1 hour at room temperature, then the KCl concentration was increased to 1 M, and the extract was incubated for 30 minutes at 37°C and centrifuged 30 minutes at 300,000 g. The pelleted actin was suspended in G-buffer, dialyzed against G-buffer to remove KCl, and centrifuged for 10 minutes at 300,000 g to remove aggregates. After polymerization with 3 mM MgCl₂, actin was ultracentrifuged for 30 minutes at 300,000 g, and the final pellet was suspended in F-buffer (3 mM MgCl₂, 10 mM Tris, pH 7.5) containing 0.2 mM ATP. All centrifugations were performed at 4°C.

All experiments using actin, myosin, and HMM purified from one pair of rats were completed within 4 days, including the day when the animals were killed. For every given pair of young and old rats, the purification of actin and myosin as well as all experiments with purified proteins were performed in parallel to ensure that all conditions were identical, except for the rats' ages. For results presented in this paper, 8 pairs of rats were used.

Proteins purified from the young rat are designated "young actin" (A_y) and "young HMM" (M_y), and the proteins purified from the old rat are designated "old actin" (A_o) and "old HMM" (M_o). Due to the variability of available muscle tissue and the amount of purified proteins, in two of the paired experiments using old and young HMM and in one of the paired experiment using young and old actin, the proteins from the young rat were substituted with rabbit actin and myosin. Control experiments showed that the actin-activated ATPase rates for young rat proteins were the same as those for rabbit proteins.

Protein Concentration
Protein concentration was measured by ultraviolet absorption, assuming molar absorption coefficients of 0.53 mg ml^–1 cm^–1 for myosin at 280 nm, 0.65 mg ml^–1 cm^–1 for HMM at 280 nm, and 0.63 mg ml^–1 cm^–1 for actin at 290 nm.

ATPase Measurements
Actin-activated HMM ATPase was measured at 25°C in 3 mM MgCl₂, 10 mM Tris (pH 7.5), and 2.5 mM ATP, at a constant concentration of HMM (0.69 µM myosin heads) and with varying concentrations of actin (from 3.5 µM to 59 µM); concentration of the liberated phosphate was determined by the Fiske–Subbarow method (33). V_max and K_m of the acto-HMM ATPase were determined by fitting the data with the Michaelis-Menten equation:

where v is the measured ATPase rate and [A] is actin concentration, using the software package Origin 7 (OriginLab, Northampton, MA). The high-salt ATPase activity of myosin or HMM was measured at 25°C in 0.6 M KCl, 50 mM Tris (pH 7.5), and 10 mM EDTA (K-ATPase) or 10 mM CaCl₂ (Ca-ATPase); protein concentrations were 0.025 mg/ml (K-ATPase of myosin and HMM), 0.3 mg/ml (Ca-ATPase of HMM), and 0.4 mg/ml (Ca-ATPase of myosin). The concentration of the liberated phosphate was determined using the malachite green method (34).

Determination of Reactive Cysteine
The reactive cysteine side chains were quantitated at 1 µM HMM and 5–8 µM actin in the presence of 0.1 M NaHPO₄ (pH 8), 1 mM EDTA, 1% SDS, and 10 mM Tris (pH 7.5) by the DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) method (35,36). DTNB (0.5 mM) was added to the prepared protein samples, and after a 15-minute incubation at 25°C the absorbance was measured at 412 nm. The cysteine content (mol/mol protein) was calculated using the absorption coefficient of the free TNB anion as 14,150 M^–1cm^–1 (37). Control experiments using a standard solution of cysteine showed that buffers, including SDS, did not affect the results.

Reagents
The solutions were made in MilliQ water and degassed for 30 minutes. All buffers, ATP, and protease inhibitors were purchased from Sigma-Aldrich (St. Louis, MO).

Data Analysis
The values of V_max, K_m, K-ATPase, Ca-ATPase, and cysteine content were obtained in paired experiments, where the results for each pair of young and old rats were expressed by calculating the ratio (old rat value) / (young rat value) and the difference (young rat value) – (old rat value). After the experiments with all 8 pairs of rats were completed, the ratios were averaged and presented on the figures as mean ± SEM. The significance of the age-related changes was determined by performing a t test ( = 0.05) on the differences for all paired data. The differences were regarded as significantly different when t > t_crit.

	RESULTS

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Contractility of Single Muscle Fibers
Age-related changes in muscle contractility were determined using the semimembranosus fibers dissected from the same animals which were the source of muscles for the purification of actin and myosin. Specific tension of the fibers from the old animals (82.7 ± 2.0 kN/m²) was 77% of that of the fibers from the young animals (106.8 ± 2.8 kN/m²), and the unloaded shortening velocity V_o of the fibers from the old animals (7.5 ± 0.5 fl/s) was 78% of that of the fibers from the young animals (9.6 ± 0.4 fl/s). These results are consistent with the age-related decline in muscle function previously observed in our laboratories (4,5). To investigate the biochemical basis of this decline, we measured the ATPase activities of the purified actin and myosin.

Effects of Aging on Actin-Activated HMM ATPase
Figure 1 is representative of a paired experimental set, in which we determined V_max and K_m of the actin-activated HMM ATPase. In this case, the data from young actin and young HMM (designated A_yM_y) are compared with the data from old actin and old HMM (designated A_oM_o). In this experiment, aging results in a significant decrease in V_max, but no significant decrease in K_m (see Figure 1 legend). After this experiment was performed on all seven pairs of rats, the mean V_max for old acto-HMM (A_oM_o, 12.15 ± 0.85 s^–1), was 21% lower than that for young acto-HMM (A_yM_y, 15.25 ± 0.75 s^–1), and the t test on the paired data sets showed that this decrease was statistically significant (n = 7, t = 11.001 > t_crit = 2.447). The mean K_m for the old acto-HMM (A_oM_o, 7.51 ± 1.05 µM) was 24% lower than that for the young acto-HMM (A_yM_y, 9.90 ± 1.11 µM), and this difference was also statistically significant (n = 7, t = 3.164 > t_crit = 2.447).

View larger version (18K): [in this window] [in a new window]   Figure 1. Representative experimental set to determine Vmax (the maximum rate) and Km (actin concentration at half Vmax). The actin-activated heavy meromyosin (HMM) ATPase rate was measured at increasing concentrations of actin. The Vmax and Km were obtained by fitting the data to Equation 1. = young actin, young HMM, Vmax = 16.43 ± 1.00 s–1, Km = 7.44 ± 1.63 µM; • = old actin, old HMM, Vmax = 12.55 ± 0.62 s–1, Km = 8.98 ± 1.48 µM

View larger version (42K): [in this window] [in a new window]   Figure 2. Age-related changes in the actomyosin function are due primarily to changes in myosin. Vmax and Km for actin-activated heavy meromyosin (HMM) ATPase were determined as in Figure 1. Data were normalized to the value for young actin and young HMM, AyMy. AoMo = old actin and old HMM; AyMo = young actin and old HMM, AoMy = old actin and young HMM. *Statistically significant

View larger version (33K): [in this window] [in a new window]   Figure 3. Age-related changes in myosin high-salt ATPase experiments. Age-related changes in the ATPase activity of heavy meromyosin (HMM). K-ATPase (open bars) was determined in the presence of 0.6 M KCl, 50 mM Tris (pH 7.5), and 10 mM EDTA; Ca-ATPase (hatched bars) was determined in the presence of 0.6 M KCl, 50 mM Tris (pH 7.5), and 10 mM CaCl2. The ATPase rates for old HMM (Old) are expressed as fraction of the rates for young HMM (Young). *Statistically significant

Reactive Cysteine Content in Myosin and Actin From Young and Old Muscle
To evaluate total cysteine content in myosin's HMM and in actin, the DTNB assay was performed on proteins unfolded by treatment with 1% SDS. In control experiments using rabbit skeletal muscle HMM, this method yielded 31.4 ± 1.2 cysteines/mol of HMM, in good agreement with the 32 cysteines predicted from the amino acid sequence (10). DTNB titration of HMM from old and young rats showed an age-related decrease in free cysteine content, from 31.11 ± 0.95 moles per mol in young HMM to 28.03 ± 0.89 moles per mol in old HMM. This difference was statistically significant (n = 6, t = 5.793 > t_crit = 2.365) (Figure 4).

View larger version (32K): [in this window] [in a new window]   Figure 4. Age-related decline in the free cysteine content of heavy meromyosin (HMM), but not actin. Cysteine content (moles reacted per mole protein) was measured by the DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid) method and normalized to the young value. *Statistically significant

Control experiments demonstrated that the purification procedure did not introduce Cys modifications in actin and myosin. We performed the initial steps of the purification process (homogenization of muscle and extraction of myosin in Guba-Straub solution) in the presence of 5 mM dithiothreitol (DTT, an antioxidant specific for Cys). Neither the actin-activated ATPase activity nor the cysteine content of purified proteins was affected by this DTT treatment. Furthermore, the cysteine content of the young as well as the old HMM purified by our standard procedure (in the absence of DTT) was not affected by a subsequent reduction at 10 mM DTT, indicating that the detected decrease in free cysteine thiols is due to irreversible modifications occurring in vivo.

	DISCUSSION

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Age-Related Changes in Myosin Function and Chemical State
To perform systematic studies of age-related changes in the functional and chemical properties of actin and myosin, we have purified actin and myosin from the hamstring and quadriceps muscles of young and old animals. These muscle were chosen because the previous research in our laboratories showed that their contractility decreases with aging and that this decrease is not associated with changes in the expression of myosin heavy-chain isoforms but may be associated with chemical changes in myosin, such as cysteine oxidation (4,5,39,40). The present study has two principal findings for purified actin and myosin: (1) a significant age-related decrease in the V_max and K_m of actin-activated ATPase and (2) a significant age-related decrease of free cysteine content in myosin.

The age-related decrease in V_max (21%), detected at the level of the purified proteins, is comparable to the 22% decrease in shortening speed of muscle fibers from the same rats (see Results section) and to the 20% decrease in unloaded shortening speed of myofibrils from the semimembranosus muscle (41). The established correlation between the unloaded shortening velocity V_o of the different muscle types (e.g., fast and slow myosin isoforms) and the actomyosin ATPase (42,43) suggests that the detected decrease in V_max of the ATPase is due to changes in the properties of the myosin molecule. The analysis of the age-induced changes in acto-HMM, HMM alone, and actin alone supports the following conclusions: young actin activated old HMM (A_yM_o) to a lower extent than young HMM (A_yM_y), whereas there was no significant difference in the activation of young HMM by young or old actin. There is a clear difference between the observed age-induced changes in the actomyosin ATPase and the observed isoform-related changes in the actomyosin ATPase (43). The decrease in V_max for the different myosin isoforms is accompanied by an increase in K_m (43), whereas the age-induced decrease is observed in both V_max and K_m. This finding suggests that the mechanism responsible for age-induced inhibition of the ATPase is due to some unique molecular change in myosin that is distinct from changes defining the different myosin isoforms. The age-related functional changes are probably due to oxidative modifications, as will be discussed.

Mechanism of Age-Related Changes in the Actomyosin ATPase
The biochemical steps of the actomyosin ATPase cycle have been associated with distinct structural states of actin and myosin. The current models postulate that muscle contraction is generated upon the transition of the actin–myosin complex from states of weak interaction (AM·ATP, AM·ADP·P) to states of strong interaction (AM·ADP, AM) (44,45) (Figure 5). In terms of this model, the age-related decrease in V_max suggests alterations in the structural states of myosin that affect the transition from weak to strong interactions. Possible transitions include disorder-to-order transitions in the catalytic and light-chain domains of myosin or changes in the internal structure of the catalytic domain (44,46). This possibility is supported by the observed correlation between changes in V_max and mutations of specific sequences within the actin-binding region of myosin (47).

View larger version (10K): [in this window] [in a new window]   Figure 5. Age-related decrease in Vmax suggests alterations in the structural states of myosin. Actomyosin ATPase cycle, indicating: (a) weak interactions of actin (A) with myosin (M)•nucleotide complexes, M•ATP and M•ADP•Pi, where ADP and Pi are products of ATP hydrolysis, and (b) strong interaction of actin with M•ADP and M alone

Our proposed interpretation in terms of age-related changes in the molecular interactions between actin and myosin may be oversimplified, as both K_m and V_max are determined by multiple transitions between intermediates of the actomyosin ATPase cycle (28,48). Thus, the understanding of the age-related changes in the molecular interaction of actin and myosin requires further biochemical as well as structural studies. The present study has taken the first step toward this goal, by demonstrating that purified actin and myosin from aged rats reveal consistent biochemical age-related changes.

Age-Related Oxidative Modifications of Myosin
We detected oxidation of 3 of 32 cysteines (10) in HMM. This finding is comparable with the previously reported oxidation of about four cysteines per myosin in the gastrocnemius muscle of rat (23). These results support the hypothesis that cysteines are important oxidative targets in the aging process (8,9). The age-induced modifications of the myosin molecule are probably facilitated by its long half-life and age-related decrease in the turnover rate (49–51).

The observed 10% decrease in K-ATPase of HMM suggests some modification of Cys 707 and/or Cys 697. However, the lack of changes in Ca-ATPase strongly suggests that the modification is not specific for these two residues (11,12). Because oxidation of cysteines is one of the possible chemical changes in myosin that results in inhibition of the actin-activated ATPase, some of the oxidized residues could be located within or in the vicinity of the functionally relevant sequences in myosin. Possible candidates include Cys 540 and Cys 402. Cys 540 is in a region important for the strong interaction with actin. Cys 402 is of particular interest because mutation of the neighboring Arg 403 to Gln causes human heart muscle dysfunction associated with familial hypertrophic cardiomyopathy (52).

The decrease in K- and actin-activated myosin ATPases could also result from oxidative modification of sites other than cysteine. Studies on rabbit skeletal myosin (53,54) have correlated inhibition of K-ATPase with modification of Lys 84 at the interface of the catalytic and lever arm domains, and with glycation of yet unspecified lysines (55). Glycation of myosin, detected in aging rats (24), has been suggested as the mechanism for the inhibition of actin sliding on aged myosin in vitro (56,57), but localization of the glycated residues remains unknown.

Age-Related Changes in Actin
We did not detect significant age-related changes in actin's functional properties or cysteine content. However, we cannot exclude the possibility that other changes in actin occur and subsequently alter muscle function via effects on actin's interaction with proteins other than myosin. It has been shown that the oxidative modification of actin inhibits the interaction with -actinin (58). The mutation-induced changes in -actinin binding sites of actin were implicated in the mechanism of myocyte dysfunction and heart failure in dilated cardiomyopathy (59). Future experiments are needed to elucidate age-related structural and functional changes in actin.

Summary
The present work explores the biochemical basis of previously observed age-related degeneration of muscle contractility. Using purified actin and myosin, we have found a significant age-related inhibition of the actin-activated myosin ATPase, and a significant age-induced decrease in the free cysteine content of myosin. Future experiments will focus on a more thoroughly analyzing the age-related chemical changes in both actin and myosin, localizing sites of oxidative modifications, and determining their specific roles in the impairment of functional actin–myosin interactions.

	Acknowledgments

 
This work was supported by grants from the National Institutes of Health to L. T. (AG17768 and AG021626) and to D. D. T. (AR32961).

We thank Dr. Dawn Lowe for stimulating discussions; Sheng Zhong for help with dissection of muscle, measurement of force and shortening velocity V_o of fibers from the semimembranosus muscle of young and old rats, and performing SDS–polyacrylamide gel electrophoresis; and Octavian Cornea for help with preparation of the manuscript. The results were presented as a poster during the Gordon Research Conference: Biology of Aging (September 2004).

	Footnotes

 
Decision Editor: James R. Smith, PhD

Received August 25, 2004

Accepted November 16, 2004

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