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J Appl Physiol 88: 973-980, 2000;
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Vol. 88, Issue 3, 973-980, March 2000

Myofibrillar or mitochondrial creatine kinase deficiency alone does not impair mouse diaphragm isotonic function

Jon F. Watchko1, Monica J. Daood1, Bé Wieringa2, and Alan P. Koretsky3

1 Department of Pediatrics, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 2 Department of Cell Biology and Histology, University of Nijmegen, Nijmegen, The Netherlands; and 3 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Creatine kinase (CK) provides ATP buffering in skeletal muscle and is expressed as 1) cytosolic myofibrillar CK (M-CK) and 2) sarcomeric mitochondrial CK (ScCKmit) isoforms that differ in their subcellular localization. The diaphragm (Dia) expresses both M-CK and ScCKmit in abundance. We compared the power and work output of 1) control CK-sufficient (Ctl), 2) M-CK-deficient [M-CK(-/-)], 3) ScCKmit-deficient [ScCKmit(-/-)], and 4) combined M-CK/ScCKmit-deficient null mutant [CK(-/-)] Dia during repetitive isotonic activations to determine the effect of CK phenotype on Dia function. Maximum power was obtained at ~0.4 tetanic force in all groups. M-CK(-/-) and ScCKmit(-/-) Dia were able to sustain power and work output at Ctl levels during repetitive isotonic activation (75 Hz, 330-ms duration repeated each second at 0.4 tetanic force load), and the duration of sustained Dia shortening was 67 ± 4 s in M-CK(-/-), 60 ± 4 s in ScCKmit(-/-), and 62 ± 5 s in Ctl Dia. In contrast, CK(-/-) Dia power and work declined acutely and failed to sustain shortening altogether by 40 ± 6 s. We conclude that Dia power and work output are not absolutely dependent on the presence of either M-CK or ScCKmit, whereas the complete absence of CK acutely impairs Dia shortening capacity during repetitive activation.

respiratory muscle; fatigue; myosin heavy chain


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CREATINE KINASE (CK), an enzyme central to cellular high-energy phosphate metabolism, catalyzes the following reaction
PCr + MgADP<SUP>−</SUP> + H<SUP>+</SUP> ↔ Cr + MgATP<SUP>2−</SUP>
where PCr is phosphocreatine and Cr is creatine. Two distinct isoforms of CK are found in skeletal muscle: 1) the sarcomeric mitochondrial CK (ScCKmit), which is localized to the mitochondrial intermembrane space where it channels high-energy phosphates produced by oxidative phosphorylation into the cytosol as PCr (22, 27), and 2) the cytosolic myofibrillar CK (M-CK), a portion of which is localized at subcellular sites of energy utilization including the actomyosin, sarcoplasmic reticulum Ca2+, and sarcolemmal Na+-K+-ATPases (22). The high level of CK activity found in skeletal muscle ensures that, when high-energy phosphate production is necessary during repetitive contractile activity, ATP levels will be maintained at the expense of PCr. This role of CK as a temporal ATP buffer is widely accepted (13, 22).

How the activity of the two CK isoforms and by inference their cellular localization modulate muscle performance remains a source of controversy. Several studies have demonstrated that the complete absence of CK activity is associated with an immediate marked decline in skeletal muscle force generation and power output during repetitive activation (7, 17, 20, 25). The functional impact of deficiency in one or the other CK isoform was recently studied in transgenic mice in which one or the other isoform was deleted by targeted mutagenesis (7, 17, 18). In this regard, most skeletal muscles express M-CK as the predominant isoform (>95% of total CK activity) with limited ScCKmit expression (1-2% of total CK activity) (22, 24). In muscles characterized by this CK phenotype, isolated M-CK deficiency is associated with an impairment of short-term muscle force-generating capacity ("burst activity") (20, 21), whereas ScCKMit deficiency alone has no adverse functional impact (18). In contrast, the absence of M-CK in muscles that express abundant ScCKmit [e.g., the costal diaphragm (Dia) (7, 24, 25) and ventricular myocardium (16)] is not associated with an adverse impact on contractile performance (7, 16). Moreover, skeletal muscle that has been engineered to express the brain (cytosolic) isoform of CK (B-CK) instead of M-CK does not show any deficit in contractile performance despite the lack of B-CK localization to the M line of myofibrils and lower-than-control levels of CK activity (~30% of control) (14). These studies indicate that specific CK isoforms may not be important for maintaining muscle force production but rather that there is a requirement for some CK activity independent of isoform.

In the present investigation, we studied Dia from wild-type control (Ctl) and transgenic mice in which CK expression had been altered by targeted mutagenesis to determine the effects of 1) isolated M-CK deficiency [M-CK(-/-)], 2) isolated ScCKmit deficiency [ScCKmit- (-/-)], and 3) combined ScCKmit/M-CK deficiency [CK(-/-)] on Dia function during repetitive isotonic activation. The Dia is of interest in this regard because it contains ~75% MM-CK and 25% ScCKmit and no significant MB- or BB-CK. Therefore, unlike most skeletal muscle, a deletion of MM-CK leaves significant levels of ScCKmit, and, unlike ventricular myocardium, which has significant B-CK, a deletion of both M-CK and ScCKmit leaves no significant CK activity. Isotonic function was indexed by changes in the extent of muscle shortening, muscle shortening velocity, power, and work during repetitive isotonic activation. Because CK isoforms are structurally associated with sites of energy consumption [M-CK and actomyosin ATPase on the myosin heavy chain (MHC)] (22) and isotonic contractile properties are related to myosin isoform composition, we also determined the effect of CK deficiency on MHC phenotype.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were conducted on adult (90- to 110-day-old) M-CK(-/-)-deficient, ScCKmit(-/-)-deficient, CK(-/-)-double-deficient, and CK-sufficient wild-type Ctl mice all of which had a mixed genetic (C57Bl/6 × 129/Sv) background. The M-CK(-/-)-deficient, ScCKmit(-/-)-deficient, and CK(-/-)-double-deficient transgenic lines were generated by Wieringa and colleagues (17, 18, 20, 21) as previously described and confirmed by PCR analysis. Seven animals from each study group were anesthetized with pentobarbital sodium (60 mg/kg ip), and individual segments of Dia were excised for 1) in vitro isotonic contractile properties, 2) measurement of CK activity and isoenzyme fractionation, and 3) determination of MHC phenotype.

In vitro measurements. Dia muscle strips (~2 mm wide) were cut from the midcostal region of the right hemidiaphragm, with fiber attachments at the rib and central tendon left intact. The muscle segments were mounted in a vertical tissue chamber, which was constantly perfused with mammalian Ringer solution aerated with 95 %O2-5% CO2 and maintained at 37°C. The monitored PO2, PCO2, and pH were 400-460 Torr, 35-40 Torr, and 7.35-7.40, respectively. The costal margin origin of fibers was fixed by use of a vascular clamp mounted in series to a micropositioner near the base of the tissue chamber. A small piece of aluminum foil was glued to the central tendon with cyanoacrylate and then attached to the force transducer (model 300B, Cambridge Technology) via fine wire. This provided a noncompliant attachment to the force tranducer and prevented tearing of the central tendon. The muscle bundles were stimulated directly (Grass model S-88 stimulator with current amplifier) by use of monophasic rectangular pulses of cathodal current (1.0-ms duration) delivered through platinum plate electrodes placed ~1 cm apart. Muscle bundles were positioned midway between the two electrodes. To ensure supramaximal stimulation, current was increased by 50% over the current necessary to obtain peak twitch force (~250-300 mA). Muscle fiber length was adjusted incrementally by using a micropositioner until maximum isometric twitch force (Pt) responses were obtained [i.e., optimal fiber length (Lo)]. Twitch contraction (CT) and half relaxation (RT1/2) times were also determined. Maximum tetanic force (Po) was assessed by stimulating the muscle bundle at 200 Hz delivered in a 1-s train. Force and length signals of the Cambridge dual-mode servo-control module were displayed on a digital oscilloscope (model 1602, Gould), digitized at 500 Hz, and stored on a computer disk file. The stimulation paradigm and isotonic afterloaded contractions were controlled by a computer program (LabView 3.1, National Instruments).

Shortening velocities were measured at eight different afterloads (5-50% of Po) during isotonic afterloaded contractions as previously described (25). Velocities were calculated by computer from the maximum slope of the digitized length signal in the interval between 10 and 30 ms after the beginning of the isotonic shortening phase. Loads were calculated as a fraction of Po based on the force plateau measured during the isotonic contraction. The data were fitted by a modified version (1) of Hill's hyperbolic equation [(V + b)(P/Po + a/Po) = b(1 + a/Po)] by using a least-squares technique, and maximum velocity of shortening (Vmax) was calculated from the optimum a/Po and b values as Vmax = b/(a/Po). Vmax is reported in Lo per second. Power was calculated as the product of the isotonic afterload and velocity of shortening and expressed in watts per square meter. Work was calculated as the product of the isotonic afterload and extent of lengthening and expressed in joules per square meter. The maximum power and work were derived from the respective power and work curves over the range of afterloads (5-50% Po) examined.

After completion of the force-velocity measurements, the muscle was stimulated repetitively under isotonic conditions (75 Hz in trains of 330-ms duration repeated each second) at 40% Po, the load that produced maximum power across CK phenotypes and control animals. Changes in velocity and extent of Dia shortening, power, and work were determined during repetitive isotonic activation. The endurance time (in s) was calculated as the interval between the initiation of repetitive isotonic activation and the cessation of muscle shortening. Following the repetitive isotonic activation paradigm, Lo was determined, and the stimulated muscle segment was weighed. Muscle cross-sectional area (CSA) was estimated on the basis of the following formula: muscle weight (g)/[Lo (cm) × 1.056 (g/cm3)]. The estimated CSA was used to determine specific tetanic (Po /CSA) force of the muscle segments.

Determination of CK activity and isoenzyme distribution. Total CK activity was determined at 25°C by using a hexokinase/glucose-6-phosphate dehydrogenase-coupled enzyme system, which ultimately yields a reduced NADP (NADPH) proportional to CK activity (Sigma Diagnostics, St. Louis, MO) (24). For this analysis, a 5- to 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 beta -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 PCr, 1.3 units of hexokinase, and 0.5 units 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 the production of NADPH through the coupled enzyme reaction, was used to determine CK activity. Care was taken to make sure that the rate obtained linearity with the volume of homogenate added. Protein was determined by the method of Lowry et al. (9), and CK activity was expressed as micromoles per milligram protein per minute.

The CK isoenzyme phenotype was resolved electrophoretically (24). Homogenized muscle tissue (as above) was centrifuged for 20 min at 14,000 revolutions/min 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 PCr, 60 mM magnesium acetate, 60 mM glucose, 60 mM N-acetyl cysteine, 15 mM AMP, 12 mM ADP, 6 mM NAD, 10 µM diadenosine pentaphosphate, 9,000 U/l hexokinase, and 7,500 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 ultraviolet light. The BB, MB, MM, and mitochondrial CK isoforms are readily separated by this electrophoretic technique (24). To determine the relative contributions of individual isoforms to their respective total CK complements, photographs of the gels were taken and analyzed by using a scanning densitometer (GS 300, Hoefer Scientific) and densitometry software (GS 365, Hoefer Scientific) to quantify the area under individual isoform peaks.

The localization of CK isoenzymes to Dia mitochondrial and cytosolic compartments across study groups was assessed biochemically by determining the CK isoform phenotype of 1) mitochondria and 2) mitochondria-free subfractions. Mitochondria from Ctl, M-CK(-/-), ScCKmit(-/-), and CK(-/-) Dia were isolated by using the technique for skeletal muscle mitochondria isolation described by Bhattacharya and colleagues (4). Briefly, the Dia was minced in ionic incubation medium consisting of (in mM) 100 sucrose, 10 EDTA, 100 Tris · HCl, and 46 KCl, as well as 0.5% BSA and 0.02% Nagarse (EC 3.4.21.62, Sigma Chemical) at pH 7.4 and homogenized (4). The resultant homogenate was fractionated by centrifugation at 500 g for 10 min. The supernatant was centrifuged twice at 12,000 g for 10 min to obtain the mitochondrial pellet. An aliquot of this supernatant was taken for CK isoenzyme analysis on agarose gel as described above. The mitochondrial pellet was resuspended in the incubation medium, and an aliquot of the suspension was similarly assayed for CK isoenzyme phenotype on an agarose gel.

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 (8). Extracts were centrifuged and supernatants recovered and treated as follows. Electrophoresis of MHC isoforms was performed by using the method of Talmadge and Roy (19). Ten microliters of supernatant were diluted (1:10) in a low-salt buffer consisting of 1 mM EDTA and 0.1% beta -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 1:100 in SDS sample buffer [62.5 mM Tris · HCl, 2% (wt/vol) SDS, 10% glycerol, 5% (vol/vol) beta -mercaptoethanol, and 0.001% (wt/vol) bromophenol blue at pH 6.8]. The samples were boiled for 2 min and stored at -80°C.

Gels were prepared from a stock solution of 30% acrylamide containing 29.4% (wt/vol) acrylamide and 0.60% (wt/vol) N,N'-methylene-bis-acrylamide. 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 of total concentration of monomer (acrylamide + N,N '-methylene-bis-acrylamide) = 8% and stacking gels of total concentration of monomer = 4% at percentage of total monomer due to N,N '-methylene-bis-acrylamide = 2% were used. Volumes of myosin extract (1-3 µl) containing 500-1,000 ng of protein per well were loaded on the gels. Electrophoresis (275 V for 3.5 h and then 178 V for 17.5 h) was performed by using a vertical-slab gel unit (SE600, Hoefer Scientific Instruments) with Tris-glycine running buffer (19) in a cold room maintained at 4°C. Separating gels were silver stained (8). Monoclonal antibodies were used to identify the specific MHC bands (MHCslow, MHCIIa, MHCIIx, MHCIIb) separated on SDS-PAGE and included 1) antibody BF-32 to MHCslow and MHCIIa; 2) antibody SC-71 to MHCIIa; 3) BF-35 to all but MHCIIx; 4) BF-F3 to MHCIIb; and 5) RT-D9 to MHCIIx and MHCIIb (8). MHC gels were analyzed by using a scanning densitometer (GS 300, Hoefer Scientific) and densitometry software (GS 365, Hoefer Scientific) to quantify the area under individual peaks. These data were used to determine the relative contribution of individual isoforms to their respective total MHC complements.

Statistical analysis. Statistical methods [Minitab Statistical Software, PC version release 8.0, Minitab, State College, PA (15)] included a two-way ANOVA for comparisons of animal weight, Lo, CK levels, MHC isoform expression, and in vitro contractile properties across CK phenotypes. When significance across CK phenotypes was observed, post hoc analysis was performed by using the Scheffé's test. To compare changes in the velocity and extent of Dia shortening, power, and work as a function of CK phenotype and time during repetitive isotonic activations, curve fitting and ANOVA were performed, followed by post hoc analysis to identify differences at individual time points. Statistical significance was established at P < 0.05. Data are reported as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Physical characteristics and isometric contractile properties. Animal and Dia characteristics as well as Dia isometric contractile properties are presented in Table 1. Animal body weight, Dia Lo, CT, and RT1/2 were not significantly different across CK phenotypes (Table 1). Dia Po /CSA, however, was significantly lower (~17-21%) in CK(-/-) mice compared with Dia Po /CSA in Ctl, M-CK(-/-), and ScCKmit(-/-) mice (Table 1).

                              
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  		Table 1.   Physical characteristics, isometric contractile properties, and force-velocity characteristics of control, M-CK(-/-), ScCKmit(-/-), and CK(-/-) diaphragm

Isotonic contractile properties. Dia force-velocity characteristics are also shown in Table 1. All four groups manifested a hyperbolic force-velocity relationship with similar a/Po values (Table 1). Vmax, however, was significantly lower in CK(-/-) Dia compared with Ctl, M-CK(-/-), and ScCKmit(-/-) Dia (Table 1). There was no significant difference in Vmax across Ctl, M-CK(-/-), and ScCKmit(-/-) Dia.

The power-load relationship from all four CK phenotypes was parabolic and maximum power was generated at 0.4-0.5 Po across all CK phenotypes. Peak power, however, was lower in CK(-/-) mice compared with Ctl, M-CK(-/-), and ScCKmit(-/-), reflecting the lower specific force generated by CK(-/-) Dia at all loads (Table 1). Similarly, the work-to-load relationship from all four CK phenotypes was parabolic in nature and maximum work was generated at 0.4-0.5 Po across all CK phenotypes. Peak work output was lower in CK(-/-) Dia compared with Ctl, M-CK(-/-), and ScCKmit(-/-) Dia, reflecting the lower Po /CSA generated by CK(-/-) Dia at all loads (Table 1).

The velocity and extent of muscle shortening, power, and work declined significantly during repetitive isotonic activation across CK phenotypes (Fig. 1). The decline in power and work associated with repetitive isotonic activation reflected significant decreases in the velocity and extent of Dia shortening, respectively, as force was clamped at 40% Po during repetitive activation. The velocity and extent of shortening, power and work after the initial series of stimuli were significantly lower in CK(-/-) compared with Ctl, M-CK(-/-), and ScCKmit(-/-) Dia (Fig. 1). These differences were sustained throughout the period of repetitive isotonic activation. Moreover, the endurance time of CK(-/-) Dia (40 ± 6 s) was significantly less than that of Ctl (62 ± 5 s), M-CK(-/-) (67 ± 4 s), and ScCKmit(-/-) (60 ± 4 s). No significant differences were noted in velocity and extent of shortening, power, or work across Ctl, M-CK(-/-), and ScCKmit(-/-) Dia during repetitive isotonic activation.





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  		Fig. 1.   Changes in velocity of shortening (A), power (B), extent of muscle shortening (C), and work (D) of control creatine kinase (CK)-sufficient (Ctl; ), myofibrillar creatine kinase (M-CK)-deficient [M-CK(-/-); black-diamond ], sarcomeric mitochondrial CK (ScCKmit)-deficient [ScCKmit(-/-); ], and combined M-CK/ScCKmit-deficient null mutant [CK(-/-); open circle ] diaphragm (Dia) during repetitive isotonic activation. First stimulation and every other one thereafter are shown until muscle failed to shorten. Significantly lower velocities of shortening, power, length changes, and work were seen in CK(-/-) as opposed to Ctl, M-CK(-/-), and ScCKmit(-/-) Dia after initial stimulus. Interval between initiation of repetitive isotonic activation and cessation of muscle shortening, i.e., endurance time, was significantly shorter in CK(-/-) Dia. Lo, optimal fiber length.

Total CK activity and isoenzyme phenotype. Total CK activity was 801 ± 42 µmol · mg protein-1 · min-1 in Ctl Dia and was composed of ~25% ScCKmit and 75% MM-CK isoforms (Fig. 2). Total CK activity in M-CK(-/-) Dia was 147 ± 38 µmol · mg protein-1 · min-1, averaging 20% of Ctl levels. Only the ScCKmit isoform was noted on CK zymogram gels of M-CK(-/-) Dia (Fig. 2). Total CK activity in ScCKmit(-/-) Dia was 620 ± 67 µmol · mg protein-1 · min-1, averaging 77% of Ctl levels. Only the M-CK isoform was noted on CK zymogram gels of ScCKmit(-/-) Dia (Fig. 2). Neither M-CK nor ScCKmit isoform was noted on CK zymogram gels of CK(-/-) Dia (Fig. 2); total CK in CK(-/-) Dia was 8.5 ± 2.3 µmol · mg protein-1 · min-1, averaging 1.1% of Ctl levels. We surmise that the residual CK activity in CK(-/-) Dia originates from immature satellite cells and the smooth muscle lining of vasculature in this muscle.


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  		Fig. 2.   Representative agarose gels stained for creatine kinase (CK) activity demonstrate isoenzyme distribution. ScCKmit and cytosolic myofibrillar (MM-CK) isoforms in Ctl, M-CK(-/-), ScCKmit(-/-), and CK(-/-) Dia. In whole muscle homogenates, neither MM nor ScCKmit isoform was noted in CK(-/-) Dia as opposed to abundant expression of both isoforms in Ctl Dia. Whole muscle homogenates of M-CK(-/-) Dia expressed only ScCKmit isoform activity, whereas whole muscle homogenates of ScCKmit(-/-) Dia expressed only M-CK isoform activity. Mitochondrial subfraction of Ctl and M-CK(-/-) Dia contained only ScCKmit, whereas cytosolic mitochondrial-free subfraction contained only M-CK in Ctl and ScCKmit(-/-) Dia and neither M-CK nor ScCKmit in M-CK(-/-).

The mitochondrial subfraction of Ctl and M-CK(-/-) Dia contained only the ScCKmit isoform (Fig. 2) and neither M-CK nor ScCKmit in ScCKmit(-/-) Dia (Fig. 2). The cytosolic mitochondrial-free subfraction contained only M-CK in Ctl and ScCKmit Dia and neither M-CK nor ScCKmit in M-CK(-/-) Dia (Fig. 2). No M-CK or ScCKmit was detected in either the mitochondrial or mitochondrial-free supernatant of CK(-/-) Dia. We did not observe any evidence of B-CK or ubiquitous mitochondrial CK isoform expression in whole muscle homogenate, mitochondrial subfraction, or cytosolic subfraction zymogram gels of any Dia.

MHC phenotype. The MHC phenotypes of Ctl, M-CK(-/-), and ScCKmit(-/-) Dia were comparable and characterized by abundant MHCIIa and MHCIIx expression (Fig. 3). There were no significant differences in individual MHC isoform expression across study groups.


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  		Fig. 3.   Densitometric comparison of myosin heavy chain (MHC) isoform expression in Ctl, M-CK(-/-), ScCKmit(-/-), and CK(-/-) Dia. MHCslow (open bars), MHCIIa (cross-hatched bars), MHCIIx (hatched bars), and MHCIIb (solid bars) isoform content are expressed as a relative %total MHC complement.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The principal findings of this study are that 1) neither isolated M-CK nor isolated ScCKmit deficiency is associated with any greater impairment in Dia power output or work performance during repetitive isotonic activation than that observed in Ctl Dia, whereas 2) combined M-CK and ScCKmit deficiency is associated with a profound early sustained impairment of Dia isotonic function during repetitive shortening contractions. These results demonstrate that Dia isotonic function is not absolutely dependent on the presence of either M-CK or ScCKmit alone; rather, there is a requirement for some CK activity independent of isoform.

The Dia is particularly well suited to study the possible linkage between intracellular CK localization and overall muscle performance because this muscle expresses both M-CK and ScCKmit in abundance (7, 24, 25). In this respect, the Dia is similar to ventricular myocardium (5, 16) but with little detectable B-CK, making the contribution from this third CK isoform negligible. Cardiac muscle, although it expresses abundant M-CK and ScCKmit, also expresses appreciable B-CK, making it more difficult to analyze the effects of manipulating M-CK and ScCKmit levels. Moreover, under the experimental conditions used in this study, Dia ScCKmit expression was limited to the mitochondria, i.e., we did not detect any evidence of ectopic localization of ScCKmit protein in the cytosol of M-CK(-/-) Dia. Similarly, Dia M-CK expression was limited to the cytosol, i.e., we did not detect any M-CK in the mitochondrial subfraction of ScCKmit(-/-) Dia. These observations are consistent with those of Steeghs et al. (18) and van Deursen et al. (20, 21) in limb muscle, the premise that mitochondrial localization sequences are dominant, and the absence of a mitochondrial import signal in M-CK. Thus Dia from transgenic mice in which CK expression has been altered via targeted mutagenesis provides a powerful tool to examine how the activity of the two isoforms, and by inference their intracellular localization, modulates muscle performance in vitro (17, 18, 20, 21).

Previous studies on skeletal muscle that express limited ScCKmit (<5% of total CK activity), i.e., the medial gastrocnemius, indicate that isolated M-CK deficiency and combined M-CK-ScCKmit deficiency result in a failure to sustain wild-type levels of twitch and tetanic force production during the early phases of repetitive activation, whereas forces stabilized at the same level as controls during the latter stages of repetitive contraction (20, 21). In contrast, the absence of ScCKmit alone is not associated with any significant change in force generation during any stage of repetitive isometric activation compared with control muscle (18). These findings suggest that the specific absence of M-CK accounted for the impairment in function of the CK(-/-) medial gastrocnemius muscle. However, this is probably not the case as gastrocnemius muscle from M-CK(-/-) mice engineered to express B-CK evinces wild-type contractile performance despite the lack of B-CK localization to the M line of myofibrils and lower than control levels of CK activity (~30% of control) (14).

We previously demonstrated that M-CK deficiency alone was not associated with any greater impairment in Dia force-generating capacity than Ctl Dia during repetitive isometric twitch or tetanic stimulation (7). Similarly, in the current study we did not observe any greater impairment in M-CK(-/-) Dia power or work output than Ctl Dia during repetitive isotonic activation. The discordant functional impact of M-CK deficiency between the medial gastrocnemius and Dia muscles may relate to the relatively abundant expression of ScCKmit in the Dia [~25% of the total CK phenotype (7, 24, 25)] compared with medial gastrocnemius muscle (1-2% of the total CK phenotype). The ScCKmit levels in M-CK(-/-) Dia may provide sufficient ATP-buffering capacity to counter the isolated absence of M-CK. Indeed, a recent study of transgenic mouse mutants with graded reductions in M-CK activity showed a strong positive correlation between the level of total CK expression in limb muscle and the ability to maintain maximal muscle force during the initial phase of isometric activation (21). More specifically, only when total CK activity had fallen below 16-34% of control levels were marked impairments of contractile performance during repetitive activation observed (21). This finding is entirely consistent with the results of the present study and the suggestion that muscle function during repetitive activation is more dependent on total CK activity than isoform localization under the energetically more taxing condition of repetitive isotonic activations where metabolic demands would be highest (6). Indeed, the repetitive isotonic activation protocol used in the present study creates a condition that emphasizes the role of CK in meeting the energy demands of contraction, although it may not strictly mimic the normal physiological situation.

We also did not observe any greater impairment of ScCKmit(-/-) Dia power or work output than Ctl Dia during repetitive isotonic activation. This finding is comparable to that reported on ScCKmit(-/-) limb muscle in which contractile performance was similar to that of wild-type CK-sufficient muscle (18). Boehm and colleagues (5) have reported that ScCKmit(-/-) oxidative muscles (i.e., soleus and ventricular myocardium) retain their ability to couple creatine phosphorylation and oxidative metabolism. They propose that M-CK in ScCKmit(-/-) muscle relocates to the mitochondrial outer membrane and, via close proximity to porin and the adenine nucleotide translocase, preserves the coupling of oxidative respiration to CK activity (5). This speculation has yet to be confirmed but provides a possible explanation for 1) the lack of phenotypic changes and 2) the resiliency of contractile function in ScCKmit(-/-) muscle. However, we found no evidence of MM-CK in mitochondrial fractions of Dia muscle, indicating that any association of MM-CK with mitochondria must be weak.

Isometric twitch characteristics were not different across CK phenotypes. In contrast, Po/CSA was significantly lower in CK(-/-) Dia compared with CTL, M-CK(-/-), and ScCKmit(-/-) Dia. The mechanism underlying this slight reduction of Po/CSA in CK(-/-) was not addressed by the present study, but this finding is consistent with previous observations on CK(-/-) Dia (7, 25), CK(-/-) limb muscle (medial gastrocnemius; Ref. 17), and appendicular muscle from rats treated with substrate analogs of Cr (e.g., beta -guanidinoproprionic acid, a poor substrate for CK that results in diminished PCr and ATP stores) (10).

The present study was the first to examine the effect of CK deficiency on the force-velocity relationship of the Dia across M-CK(-/-)-, ScCKmit(-/-)-, and CK(-/-)-deficient phenotypes. We observed a significantly lower Vmax in CK(-/-) as opposed to other CK phenotypes. The curvature of the force-velocity relationship as quantified by a/Po, however, was not different across study groups. Vmax is highly correlated with the rate of ATP hydrolysis (actomyosin ATPase activity) by the MHC (2) and reflects the maximal cross-bridge cycling rate. In the current study, however, we observed only minor variations in MHC phenotype across CK lines that did not achieve statistical significance. Thus differences in MHC isoform expression between CK(-/-)-deficient Dia and other CK phenotypes cannot account for the lower CK(-/-) Dia Vmax. Other possible contributing factors to the lower Vmax in CK(-/-) Dia worthy of investigation include alterations in regulatory contractile protein isoforms, i.e., alkaline myosin light chain composition and troponin, and/or sarcoplasmic reticulum calcium uptake and release (17, 23).

Previous studies have reported metabolic adaptations in M-CK(-/-) and CK(-/-) skeletal muscle. Both M-CK(-/-) and CK(-/-) Dia and limb muscle evince increased oxidative capacity, glycogen content, and adenylate kinase enzyme activity (7, 20, 21). These observed adaptations in CK-deficient skeletal muscle, however, do not differ in magnitude between M-CK(-/-) and CK(-/-) Dia, suggesting that they do not account for differences in contractile performance across CK phenotypes. In contrast, ScCKmit(-/-) muscle has not been reported to evince such adaptations (18). ScCKmit(-/-) medial gastrocnemius, Dia, and ventricular myocardium do not display altered morphology, oxidative phosphorylation capacity, or MHC phenotype compared with wild-type CK-sufficient muscle (18). Whether 1) PCr production is possibly linked to glycolysis in ScCKmit(-/-) Dia or 2) the linkage of mitochondria to PCr utilization is through the metabolism of lactate formation (i.e., a reducing equivalent shuttle) in transgenic null mutant lines is unclear but worthy of future investigation.

Two different models of the CK-PCr system have been proposed in the literature. In one, often referred to as the PCr-CK "shuttle hypothesis," it is asserted that CK isoenzymes are highly compartmentalized at sites of energy production and utilization (3, 22). The diffusion of PCr-Cr and the specific localization of CK at the mitochondria, sites of glycolysis, the actomyosin ATPase, sarcoplasmic reticulum Ca2+-ATPase, and sarcolemmal Na+-K+-ATPase are envisioned to provide the optimal microenvironment for efficient substrate and product channeling and enhanced muscle performance (3, 22). In contrast, an alternative model asserts that CK-catalyzed fluxes are maintained near equilibrium in the cytosol by facilitated diffusion, i.e., the specific localization of CK isoforms is not essential for muscle performance (11, 12, 26). Although there is now considerable evidence of CK isoform localization within muscle cells (22), such localization does not necessarily indicate that CK isoforms (either ScCKmit at the mitochondria intermembrane space or M-CK localized to intracellular sites) are "segregated" from the cytosol (26). Data from the present study demonstrate that Dia function is not absolutely dependent on the presence of either M-CK or ScCKmit, but rather there is a requirement for some CK activity independent of isoform. The findings of the present study are consistent with a cytoarchitectural arrangement in Dia muscle [i.e., an elaborate mitochondrial network with relatively short physical distances between sites of ATP production and consumption similar to ventricular myocardium (16)], which contributes to the resiliency of contractile performance in the M-CK(-/-) and ScCKmit(-/-) Dia.


    ACKNOWLEDGEMENTS

The present study was supported by a Career Investigator Award from the American Lung Association (to J. F. Watchko), an American Heart Association (AHA) grant with funds contributed in part by the AHA Pennsylvania Affiliate (to A. P. Koretsky), and National Heart, Lung, and Blood Institute Grants HL-02847 and HL-40354 (to A. P. Koretsky).


    FOOTNOTES

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: J. F. Watchko, Dept. of Pediatrics, Magee-Womens Hospital, 300 Halket St., Pittsburgh, PA 15213 (E-mail: jwatchko{at}mail.magee.edu).

Received 21 June 1999; accepted in final form 1 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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