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CK has been implicated in the maintenance of skeletal muscle intracellular energy supply(1–5) by catalyzing the reversible transfer of the phosphoryl group of phosphocreatine to ADP, resulting in the generation of ATP according to the following biochemical reaction:equation

If ATP levels begin to fall during increased muscle contractile,i.e. actomyosin ATPase activity, the high level of CK found in skeletal muscle will help sustain intracellular ATP levels. This role of CK as an ATP buffer is widely accepted(1–5).

CK exists in different isoforms: three, CK-MM, CK-MB, and CK-BB are cytosolic and dimers of the M (muscle) and B (brain) subunits; a fourth isoform, CK-mitochondrial (CK-mi), is located on the outer surface of the mitochondrial inner membrane(2, 3). Adult skeletal muscle expresses almost exclusively the CK-MM homodimer in the cytosol(2).

Limited information is available regarding the postnatal expression of CK in skeletal muscle(6–10), and only one previous report has been published on CK activity in the developing respiratory musculature(10); muscles that must function immediately and optimally at birth to ensure survival. In the present study we determined the postnatal expression of CK activity and isoform phenotype(CK-MM, CK-MB, and CK-BB, CK-mi) in three respiratory muscles (DIA, IC, and EAO) and the SOL control limb muscle of the rat. Moreover, because1) CK is a key enzyme in cellular energetics(2, 3) and 2) CK activity in adult skeletal muscle is specifically related to fiber energetic demands,i.e. CK activity is greatest in fibers with intermittently very high and fluctuating energy requirements(2, 11, 12), we correlated postnatal changes in CK activity with MHC isoform phenotype. The MHC is the site of actomyosin ATPase activity, and MHC isoforms are known to differ in their actomyosin ATPase activity(13) (G. C. Sieck, unpublished observations). We hypothesized that CK activity would increase during postnatal development in a muscle-specific pattern and that the increase in CK activity would relate to an increase in muscle energetic demands as reflected by MHC isoform composition.

METHODS

Sprague-Dawley rats were used in the study, and the experiments were approved by the Magee-Womens Research Institute, Institutional Animal Care and Use Committee. Animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally) and individual DIA, IC, EAO, and SOL segments were rapidly excised and frozen in liquid nitrogen for later determination of 1) total CK activity and isoform composition, and 2) MHC phenotype. All specimens were stored at -80 °C before analysis. Muscle tissue was excised from postnatal animals at d 0 (<24 h of life) (body weight: 7.8 ± 1.0 g), d 14 (32.5 ± 3.0 g), and d 30 (114 ± 12 g), and from adult animals [>115 d (485 ± 42 g) (n = 8 for each age)]. DIA, EAO, IC, and SOL samples were obtained from each animal at each age, except for d 0 tissue where individual muscle samples from littermates were pooled within a litter, and a total of eight separate litters were used to provide the n of 8 at this age. The ages selected for study included periods representative of early postnatal development (d 0 and 14), as well as postweaning [>21 d old (d 30)], and adult time periods. The excised samples of DIA were from the midcostal region of the right hemidiaphragm; the EAO from segments originating on the seventh and eighth ribs; and the IC from parasternal, cephalic, and midthoracic interosseous intercostal spaces combined. The SOL was removed in its entirety for CK and MHC analysis.

Determination of CK activity and isoenzyme distribution. Total CK activity was determined at 25 °C using a coupled enzyme assay(14–16). More specifically, the ATP generated by the CK reaction was used in a hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system which ultimately yields a reduced NADP[NADPH] proportional to CK activity (Sigma Chemical Co. Diagnostics, St. Louis, MO). For this analysis a 5-10-mg muscle sample was homogenized for 15 s in a 1:100 (wt/vol) dilution of CK extraction buffer containing 26 mM Tris, 0.3 M sucrose, 1% NP-40, and 20 mM β-mercaptoethanol at pH 8.0. Homogenates were diluted to 1:100 in extraction buffer. Thirty-microliter aliquots of diluted homogenate were added to 1 mL of CK assay buffer at 25°C containing 130 mM KCl, 10 mM Tris (pH 7.4), 1 mM MgCl2, 2 mM AMP, 50 μM diadenosine pentaphosphate, 5 mM glucose, 0.7 mM NADP, 1.5 mM ADP, 9 mM phosphocreatine, 1.3 U of hexokinase, and 0.5 U of glucose-6-phosphate dehydrogenase. AMP and diadenosine pentaphosphate were included to inhibit adenylate kinase from producing ATP. The rate increase in absorbance at 340 nm due to production of NADPH through the coupled enzyme reaction was used to determine CK activity. Care was taken to make sure the rate obtained changed linearly with the volume of homogenate added. Protein content was determined by the Lowry(17) method, and CK activity was expressed as micromoles/mg of protein/min.

The CK isoenzyme phenotype was resolved electrophoretically(16). Homogenized muscle tissue (as above) was centrifuged for 20 min at 14 000 rpm at 4 °C, the supernatant was diluted 1:10 in extraction buffer, and 1 μL of diluted supernatant was added to a 1% agarose gel (Ciba-Corning, Marshfield, MA). Electrophoresis was performed at 120 V for 20 min at 4 °C. CK activity was visualized by evenly spreading Cardiotrac CK isoenzyme reagent (90 mM phosphocreatine, 60 mM magnesium acetate, 60 mM glucose, 60 mM N-acetylcysteine, 15 mM AMP, 12 mM ADP, 6 mM NAD, 10 μM diadenosine pentaphosphate, 9000 U/L hexokinase, and 7500 U/L glucose-6-phosphate dehydrogenase) on the gel and incubating for 20 min at 37 °C. Production of NADPH in the gel was visualized directly with UV light. The BB, MB, MM, and mitochondrial CK isoforms were readily separated by this electrophoretic technique. To determine the relative contributions of individual isoforms to their respective total CK complements, photographs of the gels were taken and analyzed using a scanning densitometer(GS 300 Hoefer Scientific Instruments, San Francisco, CA) and densitometry software (GS 365, Hoefer Scientific) to quantify the area under individual isoform peaks.

Determination of MHC composition. Myosin for electrophoresis was prepared by scissor-mincing the muscle tissue in a high salt solution, pH 6.5, at 4 °C for 40 min(18). Extracts were centrifuged, and supernatants were recovered and treated as follows. Electrophoresis of MHC isoforms was performed using the method of Talmadgeet al.(19). Ten microliters of supernatant were diluted (1:10) in a low salt buffer consisting of 1 mM EDTA and 0.1% 2-mercaptoethanol (vol/vol) and stored overnight at 4 °C to allow precipitation of myosin filaments. The filament solution was subsequently centrifuged to form a pellet which was then dissolved in myosin sample buffer(0.5 M NaCl, 10 mM NaH2PO4) followed by dilution of 1:100 in SDS sample buffer [62.5 mM Tris-HCl, 2% (wt/vol) SDS, 10% glycerol, 5% (vol/vol) 2-mercaptoethanol, and 0.001% (wt/vol) bromphenol blue at pH 6.8](20). The samples were boiled for 2 min and stored at-80 °C.

Gels were prepared from a stock solution of 29% acrylamide containing 28.42% (wt/vol) acrylamide and 0.58% (wt/vol) Bis. Electrophoresis was performed on slabs (18 × 16 cm × 0.75 mm thick) consisting of an 11.5-cm separating gel and a 4.5-cm stacking gel. Separating gels ofT = 8% and stacking gels of T = 4% [T = total concentration of monomer (acrylamide + Bis)] at C = 2% (C= percentage of total monomer due to Bis) were used. Volumes of myosin extract(1-3 μL) containing 500-1000 ng of protein per well were loaded on the gels. Electrophoresis (275 V for 3.5 h then 178 V for 17.5 h) was performed using a vertical slab gel unit (SE600, Hoefer Scientific Instruments) with Tris/glycine running buffer(20) in a cold room maintained at 4 °C. Separating gels were silver-stained(21, 22). The identity of the MHC bands (MHC slow, MHC IIa, MHC IIx, MHC IIb, and MHC neonatal) of the DIA on SDS-PAGE gels at each stage of development has been previously determined using immunoblotting techniques(21, 22). The migration of EAO, IC, and SOL MHC isoforms was compared with those of the DIA to identify specific MHC isoforms and establish the pattern of MHC expression in these muscles. Heavy chain myosin gels were analyzed using a scanning densitometer (GS 300 Hoefer Scientific) and densitometry software (GS 365, Hoefer Scientific) to quantify the area under individual isoform peaks. These data were used to determine the relative contributions of individual isoforms to their respective total heavy chain myosin complements. The ratio of adult[(MHC slow) + (MHC IIa) + (MHC IIx) + (MHC IIb] to developmental [(MHC neonatal) + (MHC slow)] MHC isoform content was calculated for each muscle at each age. This term was chosen to index relative changes in the energetic demands of the entire MHC complement of the muscle(23)(G. C. Sieck, unpublished observations). The larger the numeric value of this term, the greater the energetic demands of the contractile proteins present. The MHC slow isoform term was included in both the numerator and denominator to avoid a numeric value of infinity in age groups where MHC neonatal was no longer expressed.

Statistical analysis. Statistical methods included a two-way analysis of variance to compare changes in the dependent variable, CK activity, as a function of muscle and postnatal age. Where significant interactions between grouping factors were detected, a Mann-WhitneyU test was also performed. The relationship between CK activity and MHC phenotype was studied using correlation and regression procedures (Minitab Statistical Software, PC Version Release 8.0, Minitab Inc., State College, PA)(24). Because MHC expression is strongly correlated with age(21, 23, 25–27), we also performed a stepwise regression analysis with CK activity as the dependent variable, and age, muscle, and individual MHC isoform content as the independent variables. Statistical significance was established at p< 0.05. Data are reported as a mean ± SD.

RESULTS

CK isoenzyme phenotype. A representative CK isoenzyme gel from the DIA at each postnatal time period is shown in Figure 1. This gel demonstrates that the DIA CK isoform phenotype was characterized by abundant CK-MM expression at each age. By densitometric analysis (Table 1), CK-MM was the major isoform expressed in the DIA, EAO, IC, and SOL at all ages. CK-MB and CK-BB isoforms were consistently observed at d 0, 14, and 30 in all muscles, but not in the adult; these isoforms comprised a small fraction (typically <10%) of the total CK isoform phenotype. Similarly, CK-mitochondrial comprised a small fraction of the total CK phenotype in all muscles except the DIA where CK-mitochondrial expression accounted for up to ≈25% of the total CK phenotype in the d 30 and adult animals.

Figure 1
figure 1

Representative agarose gel stained for CK activity that demonstrates CK isoenzyme distribution [CK-mitochondrial (Mi), CK-MM(MM), CK-MB (MB), and CK-BB (BB)] in DIA at different ages [d 0 (D-0), 14 (D-14), 30 (D-30), and adult]. The CK-MM isoform was expressed in abundance in all age groups.

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Table 1 Postnatal changes in CK phenotype
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Total CK activity. Total CK activity was lowest in the immediate newborn period and increased significantly in each muscle during postnatal maturation (Fig. 2). The absolute increase in total CK activity during postnatal development was greater in the IC and EAO as compared with their DIA and SOL counterparts. Moreover, the absolute total CK activity of d 30 and adult IC and EAO was greater than that observed in the DIA and SOL (Fig. 2).

Figure 2
figure 2

Three-dimensional plot of total CK activity expressed in micromoles/mg of protein/min on the y axis as a function of muscle and age. Total CK activity increased with postnatal development and was greatest in adult IC and EAO.

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MHC phenotype. The MHC phenotype (Table 2) of the newborn DIA, EAO, IC, and SOL was characterized by abundant expression of neonatal MHC. During early postnatal development there was a decline in neonatal MHC expression and the emergence of fast MHC species (IIa, IIx, IIb) in all muscles. By d 30, neonatal MHC was no longer observed in any muscle. The adult SOL MHC phenotype was characterized by a preponderance of MHC slow; the adult DIA by approximately equal portions of slow, type IIa, and type IIx MHC; and the adult IC and EAO, by a preponderance of type IIb MHC. The numeric value of the adult MHC isoform content (slow + IIa + IIx + IIb) to developmental MHC isoform content (slow + neonatal) ratio increased with postnatal maturation and was greatest in the adult IC and EAO(Table 2, Fig. 3).

Table 2 Postnatal changes in MHC phenotype
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Figure 3
figure 3

Three-dimensional plot of the adult-to-developmental MHC ratio on the y axis as a function of muscle and age. The adult-to-developmental MHC ratio increased with postnatal development and was greatest in adult EAO and IC.

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Relationship of CK activity with MHC phenotype. Total CK activity significantly correlated with age (r2 = 0.62,p < 0.001); MHCneo expression (r2 = 0.50,p < 0.001); MHC2B expression (r2 = 0.79,p < 0.001); and the adult MHC isoform content (slow + IIa + IIx + IIb) to developmental MHC isoform content (slow + neonatal) ratio(r2 = 0.93, p < 0.001; Fig. 4). The correlation between CK activity and the adult-to-developmental MHC phenotype ratio was best fit by a third-order polynomial(Fig. 4). Stepwise regression with CK activity as the dependent variable revealed only MHC IIb and age as significant(r2 = 0.92, p < 0.001), with MHC IIb expression alone accounting for 79% of the developmental variance in CK activity.

Figure 4
figure 4

Relationship between total CK activity expressed on they axis in micromoles/mg of protein/min and the adult-to-developmental MHC ratio, r2 = 0.93, p < 0.001. Each data point represents the mean value of the total CK activity and the adult-to-developmental MHC ratio for a given muscle (IC, EAO, DIA, or SOL) at a given age (d 0, 14, 30, or adult).

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DISCUSSION

The principal findings of this study are that CK activity increases significantly during postnatal development in skeletal muscle and that this increase relates to the energetic demands of muscle contractile proteins as reflected by MHC phenotype. Stepwise regression confirmed a strong association between MHC isoform expression and CK activity, with MHC IIb expression alone accounting for 79% of the developmental variance in CK activity.

We observed a marked increase in total CK activity between birth and adulthood in each of the study muscles. This increase was most pronounced in the EAO where activity rose from 329 μmol/mg of protein/min at birth to 2210 μmol/mg of protein/min in adulthood. Previous studies on limb muscle have documented an increase in total CK activity during postnatal maturation(6–7), and Kelly et al.(10) reported that CK activity more than doubled in the DIA from birth to adulthood, comparable in magnitude to our own DIA data. In all muscles and age groups studied, the CK-MM isoform was the predominant CK dimer expressed, a finding that is congruous with prior reports(6–9). One of these studies further demonstrated that the CK M subunit mRNA is first detected during late gestation in skeletal muscle, increasing rapidly to become the predominant form at birth(9). CK-MB, CK-BB, and CK-mitochondrial expression was limited in all muscles at each age, with the exception of the DIA where CK-mi made up to 25% of the CK phenotype in the d 30 and adult age groups. The greater CK-mi expression in the DIA as compared with the EAO, IC, and SOL is consistent with 1) reports that the activity of mitochondrially bound CK-mi correlates with muscle oxidative potential/mitochondrial volume density(28), and2) prior studies demonstrating higher oxidative enzyme activity in the DIA as opposed to the EAO(23) and the SOL(29). However, whether CK-mi activity is functionally coupled to oxidative phosphorylation remains unconfirmed(2, 30), and the physiologic role of CK-mi in DIA function has yet to be defined.

Peak CK activity was observed in adulthood in each study muscle. The absolute level of peak CK activity, however, varied across muscles, being highest in the IC and EAO. This finding is notable in light of previous reports on adult muscle demonstrating a relationship between CK expression and fiber type composition(11, 12); specifically, high CK activity in muscles comprised of predominantly fast twitch glycolytic fibers. Fast twitch glycolytic fibers express the adult fast MHC isoforms IIx and IIb in abundance(11, 12), and the IC and EAO are both characterized by their abundant type MHC IIb expression(23, 25, 27). The linkage between CK activity and fiber type composition in adult muscle likely resides in the MHC. MHC isoforms are known to vary in their actomyosin ATPase enzyme activity(13) with the highest actomyosin ATPase activities being observed in adult muscle fibers expressing MHC IIb(13); significantly lower actomyosin ATPase activities are observed in fibers expressing MHC slow(13). The findings in the current study of high CK activity in muscle expressing predominantly MHC IIb is consistent with 1) the observation that CK activity across adult muscle fiber types is greatest in those muscles with the greatest energy requirements(11–13) and 2) the assertion that CK is an important ATP buffer in striated muscle with high and fluctuating energy demands(1–4).

The novel observation of the current study is that the postnatal increase in CK activity relates to the energetic demands of the muscle contractile proteins as reflected by MHC isoform composition with MHC IIb expression accounting for a large percentage of the developmental variance in CK levels. It has previously been demonstrated that the actomyosin ATPase activity of developing muscle fibers is lower than that of adult muscle(31, 32) (G. C. Sieck, unpublished observations). In this regard, the neonatal MHC isoform is expressed transiently in skeletal muscle during development and is replaced by adult fast and slow isoforms during postnatal maturation. The DIA, EAO, IC, and SOL express MHC neonatal in abundance during the early postnatal period(21, 23, 25–27). As the expression of the neonatal MHC isoform declines and adult fast MHC isoforms(particularly MHC IIb with its high actomyosin ATPase activity) increase, the energy costs of contraction(23, 33) should increase. These increased energetic demands may necessitate a greater need for ATP buffering capacity, i.e. greater CK activity. The significant positive correlation between the ratio of adult to developmental MHC isoform content and CK activity we observed during postnatal maturation is consistent with this speculation and confirms previous reports that link skeletal muscle metabolic capacity with MHC phenotype during development(10, 23). The current study more specifically demonstrates a strong association between specific MHC isoform expression and CK activity, with MHC IIb alone accounting for 79% of the developmental variance in CK activity. Taken together, these observations are consistent with the assertion that skeletal muscle CK activity is specifically related to the energetic properties of their contractile proteins.

In summary, we conclude that CK activity increases during postnatal development in a muscle-specific fashion and relates to the energetic demands of the muscle contractile proteins as reflected by MHC isoform composition. We speculate that the role of CK as an energy buffer is greatest in muscles expressing the IIb MHC isoform.