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J Appl Physiol 88: 352-358, 2000;
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Vol. 88, Issue 1, 352-358, January 2000

Skeletal muscle Ca2+-independent kinase activity increases during either hypertrophy or running

Martin Flück1, M. Neal Waxham2, Marc T. Hamilton1,3, and Frank W. Booth1

Departments of 1 Integrative Biology and Pharmacology and of 2 Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77030; and 3 Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211-0001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Spikes in free Ca2+ initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca2+/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca2+, we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca2+-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca2+/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca2+-independent and Ca2+/CaM-dependent forms of Ca2+/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca2+-independent form of CaMKII was increased, like the Ca2+-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca2+-independent SRF kinase was increased, no alteration occurred in Ca2+/CaM-dependent SRF kinase activity. Thus any role of Ca2+-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

exercise; adaptation; serum response factor; ionized calcium/calmodulin-dependent protein kinase II


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PHENOTYPE OF ADULT SKELETAL MUSCLE is plastic. Overloading produces its hypertrophy, whereas unloading is associated with atrophy (7). On the other hand, low-intensity, high-repetition contractions enhance mitochondrial density without any marked alteration in muscle mass (7). Despite these profound effects of contractile activity in regulating the phenotype of adult skeletal muscle, little is known about the intracellular signaling mechanisms that link the biochemical and mechanical events of muscle contraction with the activation of translation and transcription (8). Intracellular free Ca2+ is one potential intracellular signal that could link altered contractile activity with changes in gene expression. For example, each twitch contraction transiently increases intracellular free Ca2+ from ~0.05-0.1 to 10-20 µM (25). In other cell types, modulations in intracellular Ca2+ have been shown to affect changes in nuclear functions such as gene expression, cell cycle, and apoptosis in non-muscle cells (see Ref. 22). Recently, Chin et al. (11) provided evidence for a model of Ca2+ signaling in skeletal muscle based on the relatively long recruitment (hours) undergone by the postural slow muscle fibers each day and the few minutes for the fast white fibers. In their model, the infrequent phasic high-amplitude Ca2+ transients in fast white fibers were proposed to be insufficient to maintain calcineurin, a Ca2+-regulated phosphatase, in its active state; thus nuclear factors of activated T cells proteins (NFAT) remain phosphorylated and are excluded from the nucleus, so that they are unable to bind to slow-fiber-specific genes to convert the muscle to a slow phenotype (11). Dunn et al. (15) next reported that cyclosporin attenuated, and FK506 prevented, overload-induced hypertrophy, implying that calcineurin is required for skeletal muscle hypertrophy. Others previously reported (14) that a long and low sustained plateau of Ca2+ stimulates NFAT through a Ca2+/calmodulin (CaM)-calineurin complex that stimulates serine/theonine phosphatase activity of calcineurin. Thus increases in Ca2+ and the phosphorylation/dephosphorylation status of protein are required for muscle hypertrophy.

A purpose of the present study was to determine whether the activity of kinases phosphorylating serum response factor (SRF) protein (designated here as SRF kinase activity) is increased in hypertrophying skeletal muscle. SRF is a transcription factor for the hypertrophy response element in the skeletal alpha -actin promoter (12). Carson et al. (10) recently showed an increased migration of SRF protein in nondenaturing gels during electrophoretic mobility shift assays (EMSA) with nuclear extracts from the hypertrophying anterior latissimus dorsi (ALD) muscle. Here we speculated that this alteration in SRF mobility could have been due to a change in the phosphorylation status of SRF, as SRF has been shown to be phosphorylated by Ca2+/CaM-dependent protein kinase (CaMK) in pheochromocytoma PC12 cells (26). Additionally, we have shown that both SRF kinase and Ca2+/CaM-dependent protein kinase II (CaMKII) activities in control rooster muscle phosphorylate Ser103 of SRF (unpublished observations). Ca2+/CaM binding to CaMKII initiates its autophosphorylation, which results in CaM trapping and initial induction of Ca2+/CaM-dependent kinase activity. Subsequent autophosphorylation transforms CaMKII into a form that is autonomous of Ca2+/CaM but which nevertheless maintains activity to phosphorylate substrates. These properties provide CaMKII with the capacity to encode Ca2+ signaling into sustained kinase activity (9). We observed an increased phosphorylation by Ca2+-independent SRF kinase activity (SRFCai kinase) with homogenates from 7-day hypertrophying skeletal muscle. As CaMKII is not the only kinase that can phosphorylate SRF (28), we next employed the peptide autocamtide-3, which is specially phosphorylated by CaMKII, and showed that Ca2+-independent CaMKII-like activity increased in hypertrophying skeletal muscle. To test for specificity of the exercise response to hypertrophying skeletal muscle, SRF kinase activity was then measured in skeletal muscle in a group of voluntary running rats. Surprisingly, an increased SRFCai kinase was found in a muscle of rats that have voluntarily run. In contrast to the static stretch model in the roosters, running involved ~3 h/day of dynamic muscle contractions that leads to a very different set of metabolic adaptations than in the hypertrophy model. This suggests that a change in SRFCai kinase alone does not provide specificity for the differential gene expression induced by these two exercise models.


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

Animal Care

Young roosters (White Leghorn, Texas A&M, College Station, TX) were received at 5-7 wk of age. They were housed in a run and received chicken chow and water ad libitum in the animal care facilities at the University of Texas Houston Heath Science Center.

Stretch overload. The left wing of roosters was loaded with a weight corresponding to 10% of their body weight for either 1.5, 7, or 13 days, as previously described by Flück et al. (17). The ALD muscle was harvested after anesthesia [subcutaneous injection of ketamine · HCl-xylazine-acepromazine mixture (100:4:6 mg/kg body wt)], snap-frozen in liquid nitrogen, and stored in sealed tubes at -80°C until use. The contralateral wing served as control.

Voluntary running. Female Sprague-Dawley rats weighing 175-200 g were provided running wheels and permitted to run daily (~10 km/night) for 2 wk before white vastus lateralis (WVL) muscles were removed under anesthesia (19). All muscles were snap-frozen in liquid nitrogen and stored at -80°C until use. These protocols were approved by the Institutional Welfare Committee, University of Texas Health Science Center at Houston, TX.

SRF Fusion Protein

A construct encoding full-length human SRF (SRF1-508) in pET-15b (Novagen) was received as a gift from Dr. R. J. Schwartz's laboratory (Baylor College of Medicine, Houston, TX). The published sequence of chicken SRF encodes only amino acids 134-508 (6, 12) and over these residues is 77% homologous to human SRF. Protein expression was induced in Escherichia coli strain BL21(DE3)pLysE (Novagen) with isopropyl beta -D-thiogalactopyranoside. The fusion protein was isolated by affinity-chromatography under denaturing conditions, according to the manufacturer's description. After dialysis [twice in 24 h at 8°C against a ~600-fold excess of BC-100 buffer (20 mM Tris · HCl, pH 7.9, 20% glycerol, 0.05% Nonident P-40, 100 mM KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride)], fusion protein was concentrated at 4°C by using Centriprep-10 tubes (Amicon), protein concentration was determined, and fusion protein was stored in aliquots at -80°C.

Isolation of Total Homogenate

Frozen ALD muscle was homogenized 3 × 20 s at 4°C in buffer (50 mM HEPES, pH 7.4, 0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM Na4P2O7 · 10H2O, 100 mM beta -glycerophosphate, 25 mM NaF, 50 µg/ml leupeptin, 50 µg/ml pepstatin, and 33 µg/ml aprotinin) using 5 ml of buffer per 700 mg of tissue with a Polytron mixer at a low setting (Kinematica) (16). Total homogenate was snap-frozen in aliquots at -80°C, and protein concentration was estimated (Biorad, DC protein assay). Coomassie blue staining of proteins analyzed by SDS-PAGE verified protein concentration and integrity of the extracted proteins.

In Vitro Kinase Assay With SRF Protein as a Substrate (Designated as SRF Kinase Activity)

Because 10-20% of SRF kinase activities were residual to particulate matter, total muscle homogenate was used in these assays. A 5-µl sample of total muscle homogenate (75 µg protein) was added to 45 µl preheated stage I mix: 11.11 mM HEPES, pH 7.4, 0.11% Tween-20, 5.56 mM MgCl2, 0.11 mM ATP, 0.023 µM [gamma -32P]ATP (3,000 Ci/mmol, Redivue, Amersham), and 5.56 µM microcysteinLR (Biomol) containing 22 ng/µl SRF1-508 (167 nmol), which was supplemented with 5 mM EGTA or 0.56 mM CaCl2/1.11 µM CaM, to assay Ca2+-independent and total Ca2+/CaM-stimulated SRF kinase activities, respectively (23). The reaction was run for indicated times at 30°C and stopped by the addition of 17 µl 4× SDS-PAGE loading buffer (200 mM Tris · HCl, pH 6.8, 40% glycerol, 8% SDS, 8% beta -mercaptoethanol). All reactions were run in duplicate, with a muscle homogenate sample added to stage I buffer without SRF fusion protein to monitor the amount of background phosphorylation. Alternatively, the CaMKII (autocamtide 2-related) inhibitory peptide (AIP) (KKALRRQEAVDAL) was added to 11 µM concentration in the stage I mix (10 µM final). After electrophoresis on 8% SDS-PAGE, gels were stained by using Coomassie blue and exposed to X-ray film (KODAK XAR5) to visualize radiolabeled SRF. Quantitative analysis of SRF kinase activities in total homogenate was determined by scintillation counting of radioactivity incorporated into SRF relative to background. Level of Ca2+/CaM-dependent SRF kinase (SRFCa2+/CaM kinase) was calculated from the difference between total Ca2+/CaM-stimulated SRF kinase and Ca2+-independent SRF kinase (SRFCai kinase).

In Vitro CaMKII Assay With Autocamtide-3 as a Substrate

The phosphorylation reaction mixture was identical to that described for SRF as substrate (above), except for the substitution of 16.7 µM autocamtide-3 (KKALHRQETVDAL; GIBCO) for SRF. According to Hanson and Schulman (21), the synthetic peptide autocamtide-3 is a specific substrate for CaMKII. The reaction was incubated for 90 s at 30°C, stopped by transferring 20 µl of the reaction mix onto a phosphocellulose filter (P81, Whatman), followed by three serial 10-min washes in 75 mM H3PO4. Incorporation of 32P into autocamtide-3 on the filters was quantified by liquid scintillation counting. All reactions were run in duplicate, with control reactions omitting the addition of autocamtide-3 to monitor (and subtract) the amount of background phosphorylation associating with the filter. Alternatively, AIP was added to a final concentration of 11.1 µM in stage I mix (10 µM final) to specifically inhibit CaMKII activity (24). Ca2+/CaM-dependent autocamtide-3 phosphorylation was calculated from the difference between maximal Ca2+/CaM-stimulated and Ca2+-independent autocamtide-3 phosphorylation (as defined in Ref. 13).

Statistical Analysis

Data from separate experiments but same treatment were pooled and analyzed by using a paired t-test (MS Excel). The mean SE and P values (of paired 2-tail t-test) were imported into Sigmaplot software (Jandel) and plotted accordingly. Significance level selected was 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Overload Induced a Rapid Enlargement of Rooster ALD Skeletal Muscle

Loading one wing of the rooster with 10% of its body weight stretched the ALD muscle, thus activating enlargement. After 1.5, 7, and 13 days of stretch overload, total protein per stretched ALD muscle increased by 35 ± 12, 122 ± 10, and 191 ± 19%, respectively, relative to the contralateral control muscle (n = 7). Our laboratory has reported similar growth rates for this procedure in an earlier experiment (17).

SRF Phosphorylation by Skeletal Muscle Homogenates

SRF was phosphorylated by rooster ALD muscle homogenate independent of the presence of Ca2+ in vitro (lane 2; Fig. 1A). We designated this activity for the purposes of this paper as SRFCai kinase activity. Addition of Ca2+/CaM (lane 3; Fig. 1A) further stimulated SRF phosphorylation by homogenate from rooster ALD skeletal muscle, and this additional activity was designated as SRFCa/CaM kinase activity. Phosphate incorporation by ALD skeletal muscle homogenate into SRF under Ca2+-independent and total Ca2+/CaM-stimulated conditions was 0.12 ± 0.01 and 0.33 ± 0.01 mol Pi/mol SRF, respectively, after 20 min of reaction at 30°C (Fig. 1B; n = 3 roosters). Thus SRFCa/CaM kinase activity was higher in ALD muscle homogenates than was SRFCai kinase. Extending the reaction time from 5 to 20 min only increased phosphate incorporation into SRF protein by 9% in the presence of Ca2+/CaM, but increased SRF phosphorylation 200% under Ca2+-independent conditions (Fig. 1B). These results on the saturation of SRF phosphorylation suggested that the number of phosphorylation sites on SRF is limited and that phosphorylation of SRF, due to SRFCa/CaM and SRFCai kinase in vitro, is best resolved at 5 min of reaction time.


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  		Fig. 1.   Autoradiogram from SDS-gel analyzing reactions of Ca2+-independent and Ca2+/calmodulin (CaM)-dependent phosphorylation of rooster anterior latissimus dorsi (ALD) muscle homogenates. A: homogenates of rooster ALD muscle were incubated without (lane 1) or with (lanes 2-4) serum response factor (SRF) SRF1-508 in the presence of Ca2+/CaM (lanes 1 and 4), Ca2+ (lane 2), or EGTA (lane 3) under standard phosphorylation reaction conditions (see MATERIALS AND METHODS). B: homogenates of rat white vastus lateralis muscle were incubated without (lane 1) or with (lanes 2-3) SFR1-508 in presence of Ca2+/CaM (lanes 1 and 3) or EGTA (lane 2) under standard phosphorylation reaction conditions (see MATERIALS AND METHODS). C: time course (0.1, 5, and 20 min) of SRF1-508 protein phosphorylation by ALD homogenates under Ca2+-independent (SRFCai kinase; black-diamond ) and Ca2+/CaM-stimulated (SRFCa/CaM kinase; ) conditions. Data are reported as mol Pi incorporated per mol of SRF. Values are means ± SE (n = 3 roosters).

Ca2+-Independent Phosphorylation of SRF Is Increased in Hypertrophied Rooster Skeletal Muscle

SRFCai kinase activity was increased by 47 ± 9% on the 7th day of overloading the ALD muscle (Fig. 2A; n = 7 roosters/group). No significant change in SRFCa/CaM kinase activity was denoted.


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  		Fig. 2.   SRF kinase activity in rooster ALD muscle during stretch overload (A) and in white vastus lateralis muscle after voluntary running by rats (B). SRFCai kinase and Ca2+/CaM-dependent SRF protein kinase (SRFCa/CaM kinase) phosphorylation of SRF by using homogenates from control (hatched bars) and 7-day stretch-overloaded (solid bars in A; n = 7) and voluntarily run (solid bars in B; n = 8) muscles are shown. Values are means ± SE. * P < 0.05 from control group in SRFCai kinase activity.

Ca2+-Independent SRF Kinase Activity Is Also Increased in WVL Muscle of Voluntarily Run Rats

To determine the exercise specificity of the increased SRFCai kinase activity in hypertrophied rooster ALD muscle, the WVL muscle from voluntarily run rats was tested. Similar to the observations with rooster ALD homogenate, SRF was stochiometrically phosphorylated by rat WVL muscle homogenate (data not shown). After 20 min of reaction at 30°C, phosphate incorporation into SRF under Ca2+-independent and total Ca2+/CaM-stimulated conditions with WVL homogenates was 0.29 and 0.65 mol Pi/mol SRF, respectively. SRFCai kinase increased by 43 ± 9% in the WVL muscle of rats permitted voluntary running exercise for 2 wk (Fig. 2B; n = 8). On the other hand, SRFCa/CaM kinase activity was not significantly altered after 14 days of voluntary running (Fig. 2B), similar to the lack of change denoted with hypertrophy (Fig. 2A).

Ca2+-Independent (Autonomous) CaMKII-Like Activity as Determined With Autocamtide-3 as a Substrate Is Increased in Overloaded ALD Muscle

As SRF is phosphorylated by more than one kinase, we next tested whether overload would induce a correlated increase in CaMKII-like activity. Autocamtide-3 is specifically phosphorylated by CaMKII, and thus its phosphorylation can serve as an index of CaMKII-like activity. Ca2+/CaM-dependent autocamtide-3 phosphorylation (Fig. 3B) was ~10-fold greater than Ca2+-independent phosphorylation (Fig. 3A; n = 7 roosters/group) of autocamtide-3. Ca2+/CaM-independent phosphorylation of autocamtide-3 by ALD muscle homogenates increased by 52 ± 6, 46 ± 6, and 46 ± 6% after 1.5, 7, and 13 days of stretch overload, respectively (Fig. 3A). Ca2+/CaM-dependent autocamtide-3 phosphorylation, however, was not altered by the overload (Fig. 3B; n = 7 roosters/group). We confirmed that the majority of autocamtide-3 phosphorylation in these assays was due to CaMKII-like activity by including the CaMKII-inhibitor AIP (AIP inhibited both independent and Ca2+/CaM-dependent autocamtide-3 phosphorylation in control muscles by 87% and in stretched muscles by 84-95%; data not shown). Thus Ca2+-independent CaMKII, but not CaMKII-like activity is enhanced during overload-induced enlargement of the ALD muscle.


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  		Fig. 3.   Ca2+/CaM-dependent kinase II (CaMKII) activity of rooster ALD muscle homogenates determined with autocamtide-3 as a substrate. Ca2+-independent (A) and Ca2+/CaM-dependent (B) phosphorylation of the specific CaMKII substrate autocamtide-3 by total homogenates from ALD muscle of control (hatched bars) and stretch-overloaded (solid bars) groups as a function of duration of stretch-overload. Values are means ± SE; n = 7 roosters/group. * P < 0.05 from control group.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sherwood et al. (27) recently wrote that, to determine the underlying molecular mechanisms, through which contraction causes the changes in the phenotype of skeletal muscle, the signaling molecules that convert the mechanical/biochemical contraction stimulus into intracellular responses must be defined. Attractive candidates for such signaling molecules would be factors related to the initiation of contraction (e.g., action potential or free Ca2+), or as a by-product of contraction (e.g., substrate flux, heat, ADP, oxidative stress, or mechanical sensing) (29). A further challenge is to associate one of the many now-being-reported alterations in a contractile signaling molecule to a given transcription factor in a hypertrophying skeletal muscle of a living animal. Recently, the ability of numerous protein kinases have been reported to increase with enhanced contractile activity. For example, increases in the activities of c-Jun-NH2-terminal kinase (3), mitogen-activated protein kinase (4), mitogen-activated protein kinase kinase (4), p90RSK (3), and Raf-1 (4) occur with low-intensity, long-duration contractions. Whereas p70S6k was not increased with low-intensity, long-duration contractile activity, it did increase with loading exercise of high intensity and short duration (5), emphasizing the potential uniqueness of signaling alterations. Nonetheless, to our knowledge, in a living animal, these changes in protein kinase activities have yet to be associated with a transcription factor that had previously been shown to interact with a known exercise-response element of a gene for a protein, the level of which is altered by exercise. In the present experiments, we showed an increase in the activity of kinases that phosphorylate SRF protein, a transcription factor for which mobility in EMSA is altered in nuclear extracts from hypertrophying ALD muscle of roosters (10). SRF binds as homo/heterodimer to the hypertrophy-response element (SRE1) of the chicken skeletal alpha -actin promoter in living animals. The observed increase in Ca2+-independent CaMKII activity in hypertrophied skeletal muscle implies that the Ca2+-dependent form of CaMKII had to be increased (albeit never detected) because transient activation of the Ca2+-dependent form of CaMKII is required to produce the Ca2+-independent form of CaMKII. The increased Ca2+-independent CaMKII-like activity in hypertrophied skeletal muscle also suggests a potential association between the activation of a Ca2+-independent protein kinase and the hypertrophy phenotype, possibly through the hypothesized signaling pathway to the transcription factor (SRF) (Fig. 4).


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  		Fig. 4.   A hypothetical signaling pathway whereby mechanical loading leads to an increased transactivation of the skeletal alpha -actin promoter and eventual hypertrophy of skeletal muscle. A prolonged increase in intracellular free Ca2+ is proposed to increase both Ca2+-dependent SRF kinase and Ca2+-dependent protein kinase II activities in overloaded skeletal muscle of roosters. CaMKII has been shown to phosphorylate SRF in cultured neural cells (26). We have found that SRF protein concentration and skeletal alpha -actin promoter as well as skeletal alpha -actin mRNA increase in overloaded ALD muscle of roosters (see Refs. 7, 10, and 17). Question marks indicate that these steps have not been measured in skeletal muscle of living animals. Horizontal arrow with a question mark indicates that this is a hypothetical cross-talk from another signaling pathway that is downstream of Ca2+-dependent SRF kinase and Ca2+-dependent protein kinase II activities.

Overload increased Ca2+-independent CaMKII-like activity in skeletal muscle, whereas Ca2+/CaM-dependent like-activity was not changed with hypertrophy. This implies that hypertrophy stimulated transition of CaMKII activity into the Ca2+-independent (autonomous) state. Generation of autonomous forms of CaMKII is dependent on autophosphorylation of regulatory sites subsequent to sustained interaction of CaMKII with Ca2+/CaM (9). Autophosphorylation and generation of autonomous CaMKII activity is rapidly induced after augmentation in intracellular Ca2+ in culture (1). Thus elevated Ca2+-independent CaMKII-like activity in hypertrophied skeletal muscle most likely reflects previous activation of CaMKII due to increased interaction with Ca2+/CaM.

Related to our findings is the recent report of Antipenko et al. (2), who found that CaM concentration in the extensor digitorum longus muscle was increased by the second day of its continuous electrical stimulation. Furthermore, the CaM membrane cytosolic ratio decreased as early as 12 h after the onset of electrical stimulation, which Antipenko et al. interpreted to indicate an intracellular CaM redistribution within the muscle cell. In addition, total Ca2+-CaM-dependent protein kinase activity increased after 2 days of continuous stimulation, which Antipenko et al. interpreted as being consistent with the increased level of total and free Ca2+ in stimulated muscle. However, measurements by Antipenko et al. did not include differentiation between the Ca2+-independent and Ca2+/CaM-dependent protein kinase. Our observation of the increased Ca2+-independent protein kinase activity with both substrates SRF and autocamtide-3 provides support for Antipenko and associates' speculation regarding a prolonged response from the exercise-induced rises in free intracellular Ca2+.

There was a sustained increase in SRF kinase activity in trained WVL muscle. The rationale for using running in addition to stretch overload was to test whether endurance training (repetitive low-force contractions) caused a different response to exercise in the various kinase activities studied. As an aerobic exercise model, we selected the WVL muscle from rats that had undergone 2 wk of voluntary running because a previous study (19) had found adaptations indicative of endurance training. Approximately threefold increases in lipoprotein lipase activity, protein, and mRNA were observed in the WVL muscle in 2-wk voluntary run rats (19). Rats were killed ~2-4 h after the dark cycle was ended, during which 95% of their voluntary running activity occurred. Three potential interpretations could explain our observation that Ca2+-independent SRF kinase activity increased in both hypertrophy and running. First, it is possible that a species or gender difference (rooster vs. female rat) is responsible. Second, the response could be fiber-type specific (slow ALD vs. WVL muscle). Finally, it is conceivable that a differential expression of cofactors or downstream events of this kinase could occur to provide specificity to the different phenotypes from hypertrophy and running. Nevertheless, the sustained increase in SRF kinase activity emphasizes a potential maintenance of Ca2+ signaling long after an exercise period ends. We believe that this study on Ca2+-independent and Ca2+-dependent kinase activities will be an important part of understanding how skeletal muscle adapts to both aerobic and resistance training.


    ACKNOWLEDGEMENTS

Support was provided by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393 (to F. W. Booth), the Swiss National Foundation (to M. Flück), the National Space Biomedical Institute (to F. W. Booth), and National Heart, Lung, and Blood Institute Grant HL-57367 (to M. T. Hamilton).


    FOOTNOTES

Present address of M. Flück: M. E. Müller-Institut für Biomechanik, Universität Bern, Murtenstrasse 35, Postfach 30, CH-3010 Bern, Switzerland.

Original submission in response to a special call for papers on "Molecular and Cellular Basis of Exercise Adaptations."

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: F. W. Booth. Dept. of Veterinary Biomedical Sciences, E102 Vet. Med. Bldg., 1600 E. Rollins, Columbia, MO 65211 (E-mail: boothf{at}missouri.edu).

Received 25 August 1999; accepted in final form 20 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abraham, S. T., H. Benscoter, C. M. Schworer, and H. A. Singer. In situ dependence for activation of Ca2+/CaM-dependent protein kinase II in vascular smooth muscle cells. J. Biol. Chem. 271: 2506-2513, 1996[Abstract/Free Full Text].

2.   Antipenko, A., J. A. Frias, J. Parra, J. A. Cadefau, and R. Cusso. Effect of chronic electrostimulation of rabbit skeletal muscle on CaM level and protein kinase activity. Int. J. Biochem. Cell Biol. 31: 303-310, 1999[Web of Science][Medline].

3.   Aronson, D., M. D. Boppart, S. D. Dufresne, R. A. Fielding, and L. J. Goodyear. Exercise stimulates c-Jun NH2 kinase activity and C-Jun transcriptional activity in human skeletal muscle. Biochem. Biophys. Res. Commun. 251: 106-110, 1998[Web of Science][Medline].

4.   Aronson, D., M. A. Violan, S. D. Dufresne, D. Zangen, R. A. Fielding, and L. J. Goodyear. Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle. J. Clin. Invest. 99: 1251-1257, 1997[Web of Science][Medline].

5.   Baar, K., and K. Esser. Phosphorylation of p70S6K correlates with increased skeletal muscle mass following resistance exercise. Am. J. Physiol. Cell Physiol. 276: C120-C127, 1999[Abstract/Free Full Text].

6.   Belaguli, N. S., L. A. Schildmeyer, and R. J. Schwartz. Organization and myogenic restricted expression of the murine serum response factor gene. A role for autoregulation. J. Biol. Chem. 272: 18222-18231, 1997[Abstract/Free Full Text].

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8.   Booth, F. W., B. S. Tseng, M. Flück, and J. A. Carson. Molecular and cellular adaptation of muscle in response to physical training. Acta Physiol. Scand. 162: 343-350, 1998[Web of Science][Medline].

9.   Braun, A. P., and H. Schulman. The multifunctional calcium/CaM-dependent protein kinase: from form to function. Annu. Rev. Physiol. 57: 417-445, 1995[Web of Science][Medline].

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