(Received for publication, May 9, 1995)
From the
The regulation of contractile activity in mice bearing a null mutation of the M-isoform of creatine kinase gene, has been investigated in tissue extracts and Triton X-100-treated preparations of ventricular, soleus, and gastrocnemius muscles of control and transgenic mice. Skinned fiber experiments did not evidence any statistical difference in the maximal force or the calcium sensitivity of either muscle type. Rigor tension development at a low MgATP concentration was greatly influenced by phosphocreatine in control but not in transgenic mice as should be expected. In calcium-activated ventricular preparations, although the force developed by each cross-bridge was the same in control and transgenic animals, the rate constant of tension changes appeared to be markedly slowed in transgenic animals. As the ventricular isomyosin pattern was not altered, we suggested that, in transgenic animals, cross-bridge cycling was hindered by a local decrease in the MgATP to MgADP ratio, due to lack of a local MgATP regenerating system. Myokinase activity was not significantly changed while activities of pyruvate kinase or glyceraldehyde-3-phosphate dehydrogenase were found to be increased in transgenic animals. These results show that no fundamental remodelling occurs in myofibrils of transgenic animals but that important adaptations modify the bioenergetic pathways including glycolytic metabolism.
Creatine kinase (CK) ()is an important enzyme
catalyzing the reversible transfer of a phosphate moiety between ATP
and creatine. A major part of muscle creatine kinase exists as dimers
composed of two subunits, M and B, giving three isoenzymes, MM, BB, and
MB. In addition, there is a fourth isoenzyme in the mitochondria
(mitochondrial CK), which differs biochemically and immunochemically
from the cytosolic forms and can present octameric and dimeric
structures (Wyss et al., 1992). Studies with subcellular
fractionation or histochemical localization have revealed that CK
isoenzymes are present in cytosol or bound to intracellular structures.
M-CK has been found in myofibrils and described as a structural protein
of the M-band participating in the connections between myosin filaments
inside muscle fibers (Wallimann et al., 1977). Additional
binding sites have been described on actin filament (Wegmann et
al., 1992) or on the entire myosin filament (Otsu et al.,
1989). M-CK activity in myofibrils is as high as about 2 IU/mg of
protein in skeletal and ventricular muscles and represents 5% of total
CK activity in fast-twitch muscle compared to 23% in ventricular muscle
(Wallimann et al., 1977; Ventura-Clapier et al.,
1987b; for review see Ventura-Clapier et al.(1994)). M-CK has
been shown to be functionally coupled to myosin ATPase. That means that
myosin ATPase preferentially uses ATP supplied by creatine kinase
rather than cytosolic ATP (Bessman et al., 1980; Saks et
al., 1984). Myofibrillar CK can rephosphorylate all of the ADP
produced by myosin ATPase (Saks et al., 1976; Wallimann et
al., 1984; Arrio-Dupont et al., 1992) and can provide
enough energy for maximal force and normal kinetics even in the absence
of MgATP, at the expense of phosphocreatine (PCr) (Ventura-Clapier et al., 1987a; for review see Ventura-Clapier et
al.(1994)).
The creatine kinase/phosphocreatine system is considered to fulfill important roles in the energy metabolism of skeletal and cardiac muscles (for reviews see Wallimann et al.(1992), Wyss et al.(1992), and Saks et al.(1994)). In skeletal muscles, activity pattern determines fiber type and metabolic profile. Fast-twitch (white) muscles exhibiting rapid and brief activity patterns are mainly glycolytic and contain high amounts of PCr and CK (Iyengar, 1984; Yamashita and Yoshioka, 1991). By contrast, slow-twitch (red) skeletal muscle or cardiac muscle, exhibits prolonged and sustained activity associated with well developed oxidative metabolism and relatively low contents of PCr and CK. The organization of the CK system appears different in these two kinds of muscles; there is an abundance in cytosol of the muscle form of creatine kinase (M-CK) enzyme in fast-twitch muscle and compartmentation of the mitochondrial and M- isoenzymes in slow-twitch muscle and ventricle.
Functional consequences and adaptive
strategies observed in animal models of long term deficiency in the CK
system may give insights into the physiological role of this system in
different muscle types. Long term alterations in the creatine
kinase/phosphocreatine system have been developed by feeding animals
with slowly metabolized analogues of creatine (-guanidinopropionic
acid,
-GPA). These animals exhibit decreased PCr and ATP
concentrations in cardiac and skeletal muscles (Shoubridge and Radda,
1984; Kapelko et al., 1988; Zweier et al., 1991). In
addition, a clear cardiac hypertrophy and isoenzyme shift from fast
V
to slow V
myosin have been observed (Mekhfi et al., 1990). Adaptive strategy in the heart is directed
toward an increase in the number of contractile units together with an
increased efficiency of each unit to respond to decreased metabolic
fluxes. In the same animal model, skeletal muscles exhibited an
enhanced oxidative metabolism and an isomyosin shift from fast- to
slow-type isomyosins (Shoubridge et al., 1985; Moerland et
al., 1989).
More recently, a mouse line bearing a null mutation of M-CK has been developed (van Deursen et al., 1993). Genetic M-CK knocking out is a unique model of complete isoenzyme-specific CK deficiency in contrast to GPA feeding, a model of substrate deficiency. Another important difference between these two models is that mutant muscles keep mitochondrial and, in principle, brain isoforms of CK which could participate in energy metabolic pathways. It was shown that M-CK deficiency does not lead to compensatory overexpression of other CK isoenzymes. Muscles from these mice, which do not express the muscle form of CK, are able to use PCr but lack the ability to perform burst activity. In order to get insights into the functional characteristics and possible adaptational processes at the level of myofibrils in these transgenic mice, we characterized intrinsic mechanical properties of ventricular, soleus, and gastrocnemius muscles using the skinned fiber technique which allows us to investigate myofibrillar properties without interference with cytosolic substrate and ion changes. The results show that intrinsic mechanical capacities and calcium sensitivity were maintained in transgenic animals, although skinned fibers were not able to utilize PCr. However, contractile kinetics were markedly slowed down despite an unchanged myosin isoenzyme profile. In addition, the energy supply pattern was changed since glycolytic capacity seemed to increase in fast-twitch muscle as well as in ventricular muscle.
Six control adult
female mice C57BL/6 and 5 adult transgenic mice were anesthetized with
an intraperitoneal injection of pentobarbitone according to the
recommendations of the Institutional Animal Care Committee (INSERM,
Paris, France) and weighed. While under anesthetics, animals were
exsanguinated, and various organs were isolated and weighed. Heart,
gastrocnemius, and soleus muscles were placed in a modified Krebs
solution containing (mM): NaCl, 118; KCl, 4.7;
NaHCO, 25; KH
PO
, 1.2; and
MgSO
, 1.2.
Organ samples were frozen for further
analysis. Other samples were minced with scissors, placed into cold
solution (50 mg wet weight per 1 ml) containing (mM):
KHPO
, 100 (pH 8.7); EGTA, 1; N-acetyl
cysteine, 15; and homogenized in a Ultra-Turrax homogenizer. Tissue
homogenates were incubated for 60 min at 0 °C for complete
extraction of CK and other enzymes, centrifuged at 13,000
g for 20 min, and the supernatant was used for determination of CK
and myokinase and frozen.
Each fiber bundle was submitted to a set of
solutions of increasing calcium concentrations (see ``Materials
and Methods''), and pCa/tension relations were calculated
according to the Hill equation. Mean pCa for half-maximal
activation (pCa) and n values are
reported in Table 2. No significant change in calcium sensitivity
could be detected except a small increase in Hill coefficient in
gastrocnemius muscle.
It is known, however, that inactivation of myofibrillar CK, either by inhibition or in the absence of PCr, leads to a change in the calcium/tension relationship. This was confirmed in cardiac fibers of control mice (Fig. 1) where omission of PCr in activating solution led to an increase in calcium sensitivity from 5.68 ± 0.03 to 5.98 ± 0.03 (n = 4, p < 0.001).
Figure 1:
Original tension recording of the
responses of a control mouse skinned ventricular fiber to an increase
in calcium concentration in the presence or in the absence of PCr. Letters represent different pCa values: a,
9; b, 6.25; c, 6.125; d, 6; e,
5.875; f, 5.75; g, 5.625; h, 5.5; i, 5.375; j, 4.5. Diameter of the fiber was 210
µm. Resting tension was 4.88 mN/mm. T
was 30.1 mN/mm
. pCa
and Hill coefficient were, respectively, 5.72
and 2.04 in the presence and 6.01 and 1.51 in the absence of PCr.
Notice that active tension developed for lower calcium concentrations
in the absence of PCr.
Figure 2:
Graphs showing relative pMgATP/rigor
tension relations with or without PCr, obtained in ventricular (V), soleus (S), and gastrocnemius (G)
skinned fibers from control (continuous lines) and mutant mice (dashed lines). pMgATP versus rigor tension
relations were calculated and plotted according to the Hill equation T = K/(K +
[MgATP]), where T is relative
tension, K is a constant, and n the Hill
coefficient. Each curve was drawn using the means of the n values and pMgATP for half-maximal tension (pMgATP
) calculated for each fiber and averaged
in Table 2. Arrows indicate CK efficacy values in
control animals. Addition of PCr (solid lines) was able to
shift the pMgATP/tension relation of all muscles of control
animals but not of mutant animals.
Figure 3: Mechanical characteristics of ventricular skinned fibers from control and transgenic mice measured using the quick length change technique. k is the mean of the rate constants of tension changes following stretches of different amplitudes; TR is the level of tension recovery following stretches expressed in percent of tension changes; F/S is the force to stiffness ratio determined for each fiber. Only the rate constant of tension changes was decreased in mutant mice while the other properties were preserved.
Indeed, inhibition of myofibrillar CK
slows down tension kinetics in skinned rat cardiac muscle, probably due
to local accumulation of protons and MgADP (Ventura-Clapier et
al., 1987b). This was also observed in mouse heart where
withdrawal of PCr decreased the rate constant from 103 ± 17
s to 38 ± 4 s
(n = 4, p < 0.05).
Two types of gel electrophoresis were used to analyze the content in myosin isoforms of the cardiac and skeletal muscles, respectively. The cardiac ventricular myosins are best separated by gel electrophoresis under nondissociating conditions (Lompréet al., 1981), as shown here in a control rat heart, which displayed the three isoforms V1, V2, and V3 (Fig. 4a). Both control and transgenic mouse ventricles displayed only the V1 isoform.
Figure 4: A, gel electrophoresis of cardiac ventricular native myosin. a, control mouse ventricle. b, transgenic mouse ventricle. c, rat ventricle. V1, V2, and V3 designate the three types of ventricular myosin. B, myosin heavy chains of soleus and gastrocnemius muscles. a, control mouse soleus. b, transgenic mouse soleus. c, control mouse gastrocnemius. d, transgenic mouse gastrocnemius. 2A, 2X, 2B, and 1 designate the four types of skeletal muscle myosin heavy chains.
To analyze the myosin isoform content in the skeletal muscles, gel electrophoresis in the presence of SDS allowed the separation of the slow type isoform 1 and the three fast type isoforms 2 (Fig. 4b). The only transgenic mouse soleus muscle contained the 2A and the 1 isoforms in the same proportions, 35% and 65%, respectively, as the control muscle. The gastrocnemius muscles mainly contained the 2B isoform, 94 ± 2% (n = 4) in the control mice and 89 ± 2% (n = 4) in the transgenic mice; the difference was not significant. The remaining myosin was distributed between a trace of the slow-type 1 isoform and the two other fast-type 2A and 2X isoforms.
In this study, attempts were made to characterize the intrinsic properties of cardiac and skeletal myofibrils of mice bearing a null mutation for the M-form of CK. Skinned fiber technique was used to destroy cellular membranes, while keeping the cellular architecture intact, so that intrinsic mechanical properties of the myofibrillar network, in a definite medium surrounding myofibrils, could be investigated. The results showed that maximal force and stiffness characteristics were not altered while kinetics of force changes assessed in ventricular tissue were markedly reduced despite an unchanged isomyosin profile. Sensitivity to added ATP was not altered, while addition of PCr was without effect in mutants, suggesting no unknown route for PCr utilization inside myofibrils. Increased glycolytic activity could be one possible adaptational way to control the ATP/ADP ratio inside myofibrils devoid of bound CK during contraction.
Muscle contraction is the result of cyclic association
between the thin and thick filaments resulting in the relative sliding
of these filaments past each other when muscle is allowed to shorten,
or resulting in force development in isometric conditions. This
mechanical interaction or cross-bridge cycling is coupled to the
hydrolysis of ATP to ADP by myosin ATPase located on the thick filament
and regulated by the binding of calcium to the troponin complex of the
thin filament. The products of ATP hydrolysis are released when myosin
is attached to actin during the power stroke portion of the cycle, and
an increase in hydrolytic products such as ADP, inorganic phosphate,
and H is expected to influence the different steps and
thereby the power stroke. Earlier studies have shown that MgADP
increases isometric tension and calcium sensitivity and decreases
maximal velocity of shortening or kinetics of force development (Brandt et al., 1982; Cooke and Pate, 1985; Ventura-Clapier et
al., 1987a; Hoar et al., 1987). ADP detachment is
considered to be the rate-limiting step in crossbridge detachment and
for the overall cross-bridge cycle (Siemankowski et al.,
1985). ADP accumulation may inhibit the interaction between actin and
myosin by competing with MgATP at the active site of the myosin
molecule, thus slowing down MgADP detachment and further MgATP binding
and cross-bridge detachment (for review see Ventura-Clapier et
al.(1994)).
The rate constant of tension recovery following stretches is an estimate of the kinetics of cross-bridge cycling and reflects the rate-limiting step in the cycle; it was shown to vary with myosin isoform composition as well as following alterations in concentrations of substrates or products of myosin ATPase (Ventura-Clapier et al., 1987a; Mekhfi and Ventura-Clapier, 1988; Mayoux et al., 1994). We have observed a 3-fold decrease in cross-bridge cycling rate in cardiac myofibrils of transgenic animals compared to control without any shift in myosin isoforms. A similar change was observed in control mice when PCr was omitted in the solution. Thus, this decreased rate of force changes can be attributed to changes in ATP/ADP ratio in the vicinity of myosin ATPase with a consequent product inhibition of ATPase activity. Accumulation of MgADP as a result of a lack of myofibrillar CK will induce an increase in force production and a decrease in rate of cross-bridge cycling, leading to a lower energy consumption and better economy of force production. As a consequence, the rates of force production and relaxation of the muscle twitch would be decreased. However, for cardiac muscle having cyclic activity, this would tend to increase the end-diastolic pressure and to decrease the ventricular filling, except if the intrinsic heart rate is decreased. Unfortunately, no information are as yet available concerning heart rate, developed pressure, or the force-length relationship of cardiac muscle in transgenic animals. Although the cross-bridge cycling rate of skeletal muscle could not be determined in this study, it is highly probable that tension kinetics would be slowed also. Further studies are needed to clarify the contraction kinetics of the intact muscles in these animals.
In Triton X-100-treated fibers, loosely bound enzymes are usually detached from the myofibrillar structures. In intact cells, many enzymes including glycolytic enzymes, AMP deaminases, and myokinase are bound to myofibrillar proteins, mainly to the thin filament, and may participate in MgADP/MgATP regulation in myofibrils (Maughan and Godt, 1989). Indeed, we observed in total tissue extracts of both cardiac and skeletal muscles of transgenic mice, an increase in glycolytic enzyme activities, with no increase in total myokinase activity. It is thus possible that a fraction of these enzymes is bound to myofibrils in vivo and ensures local rephosphorylation of MgADP.
When PCr was
omitted, it was clear that calcium sensitivity of control cardiac
fibers was increased. Such a result was already obtained in rat heart
(Ventura-Clapier et al., 1987a) and is due to cross-bridge
slowing and cooperative interaction between attached cross-bridges.
Surprisingly, such an increased calcium sensitivity was not observed in
soleus, gastrocnemius, or ventricular muscles of transgenic mice, and a
similar force/calcium relationship was observed in control and
transgenic muscles, suggesting that another mechanism compensated for
the increased calcium sensitivity following changes in the local
ATP/ADP ratio. Calcium sensitivity is determined by the binding of
calcium to troponin C as well as by interactions between the other
constituents of the thin filament. Calcium sensitivity of cardiac or
skeletal muscle is developmentally regulated, and the role of troponin
T isoforms is often put forward to explain changes in calcium
sensitivity in spite of unchanged troponin C expression (Solaro et
al., 1988; Nassar et al., 1991; Pan and Potter, 1992).
One may suggest that a phenotypic change in the proteins constitutive
of the thin filament will participate in maintaining constant calcium
sensitivity in these transgenic muscles. Alternatively, at least in
cardiac muscle, cAMP-mediated phosphorylation of the inhibitory unit of
troponin (troponin I) decreases the sensitivity of myofibrils for
calcium by diminishing the Ca-affinity of troponin C
(Ray and England, 1976). Phosphorylation of troponin I has been shown
to be very stable (Garvey et al., 1988). It may thus be
possible that myofibrils of transgenic mice exhibit an enhanced
phosphorylation level of contractile proteins which would decrease
calcium sensitivity and compensate for the change induced by the
altered ATP/ADP ratio inside myofibrils. Unfortunately, experimental
data in support of such a hypothesis are lacking.
The skeletal muscle function of mice deficient in muscle CK has been investigated previously (van Deursen et al., 1993). Mice lacking M-CK have lost the ability to sustain maximal force output during short periods of high work, while apparently being adapted for endurance exercise. However, cardiac function of mutant mice has not been investigated at present. To elucidate the role of the CK system in energy metabolism, other strategies designed to reduce the activity of the CK system were used, such as feeding animals with creatine analogs. This affects the creatine kinase/phosphocreatine system at the substrate site. Alternatively, acute iodoacetamide poisoning of CK has also been used (Fossel and Hoefeler, 1987; Kupriyanov et al., 1991). In these models, where the function of isolated heart was impaired, a decreased developed pressure and rate pressure product were described (Mekhfi et al., 1990; Zweier et al., 1991). Furthermore, impairment of diastolic function and a steeper rise in stiffness at increased afterloads in association with increased energy breakdown were observed (Kapelko et al., 1988; Kupriyanov et al., 1991). Even more interesting was the observation, in these models, of phenotypic conversion of fast-twitch to slow-twitch fibers in skeletal muscle together with isomyosin transitions (Moerland et al., 1989) and cardiac enlargement and increased economy of contraction by a shift from the fast isoform of myosin to the slow isoform in heart (Mekhfi et al., 1990). It could be concluded that CK/PCr system alterations induce contractile abnormalities and that alterations in metabolic state per se, may lead to changes in the expression of contractile proteins.
No obvious change in size and distribution of the three fiber type populations was observed in M-CK-deficient mice (van Deursen et al., 1993). However, M-CK-deficient type 2A and 2B fibers exhibited a clear metabolic phenotype change by elaborating an intermyofibrillar mitochondrial network, with a high number of relatively large mitochondria, the potential for aerobic energy generation being increased approximately twice (Veksler et al, 1995) explaining improved endurance performance during low intensity exercise (van Deursen et al., 1993). In addition, we showed in the companion paper that mitochondria in ventricular and soleus muscles from transgenic mice have an increased sensitivity to ADP (Veksler et al., 1995). Increase in mitochondrial content in ``glycolytic'' muscles and increased sensitivity to ADP in ``oxidative'' muscles appear to represent adaptations toward increased energy turnover via the adenylate pathway.
Absence of
marked isomyosin shift, either in skeletal or cardiac muscle in M-CK
knocked out mice is in contrast with what was observed in rat cardiac
or mice skeletal muscles following -GPA feeding (Shoubridge et
al., 1985; Moerland et al., 1989; Mekhfi et al.,
1990). In these situations, a better economy of contractile force
development was achieved by a switch from fast to slower myosin
isoforms. The reason for such a difference is not straightforward.
Despite the absence of an isomyosin shift in mouse heart following
-GPA feeding, these hearts can potentially switch totally from
fast to slow myosin as has been shown under the influence of
hypothyroidic treatment (Ng et al., 1991). The main difference
between the two models is that ATP as well as PCr contents are
preserved in the case of the M-CK mutation in comparison with
-GPA
feeding where both compounds appear to be decreased. A consequence of
this would be that the expression of proteins of the contractile
apparatus is more under the control of the concentrations of
metabolites. On the other hand, it should be borne in mind that
targeted mutations affect the animals in the early embryonic life where
the potentialities for adaptations are much larger than in the adult
animals and may have involved more integrated adaptation mechanisms.
NMR experiments showed that fluxes through CK were not detectable in
skeletal muscle of CK-deficient mice until a threshold of activity was
reached (van Deursen et al., 1993, 1994). This suggested that
bound CK fluxes are NMR invisible and only when CK activity reaches a
certain level which allows saturation of binding sites, and when
cytosolic CK appears, CK fluxes become detectable. However, contracting
muscles were still able to hydrolyze PCr which suggested that M-CK was
not the only enzyme catalyzing the transfer of PCr to ATP. In
myofibrils, PCr is actively used up by bound CK; this has been shown in
cardiac skinned fibers by the shift of the dependence of rigor tension
development toward lower ATP concentrations induced by PCr
(Ventura-Clapier and Veksler, 1994, for review see Ventura-Clapier et al.(1994)). Although we showed that such a shift was also
present in fast-twitch and slow-twitch control skeletal muscle fibers,
it was absent in skeletal as well as ventricular fibers of
M-CK-deficient mice. No unknown enzyme or other isoform of CK, retained
after skinning, was thus present and able to use PCr and regenerate ATP
inside the myofibrillar compartment. In M-CK-deficient mice, due to the
absence of local rephosphorylation, ADP should accumulate in myofibrils
and diffuse in the cytosol toward mitochondria to be rephosphorylated
either by mitochondrial CK which could work in both directions,
explaining utilization of PCr during activity, or directly through
translocase and oxidative phosphorylations. This direct route for ADP
is favored by a decreased K of
mitochondrial respiration for ADP, an increased mitochondrial network,
and mitochondrial activity and increased glycolytic capacities (van
Deursen et al., 1993; Veksler et al., 1995). Thus,
alterations in one side of the CK system, i.e. utilization
site, induces adaptations in the opposite site, i.e. synthesis
site, showing the important role of CK in coupling utilization and
consumption of energy inside muscle cells (for review see Saks et
al.(1994)).
Of primary importance to clearly understand the exact extent and limit of adaptational processes in M-CK-deficent muscles is to know the kinetics of force development and, for the heart, to which extent it will sustain normal activity and respond to adrenergic stimulation or increase in workload. A need for more classical but necessary physiological data is evident in order to infer the exact extent and limit of adaptational processes as well as the real role of the specific isoenzymes of CK in muscle cells.
The possibility of completely and selectively abolishing a given function using transgenic technology is a fantastic tool for studying the exact role of one given protein within a pathway or a function, or for life. It has been disappointing, however, since functions considered as essential for life could be suppressed without lethal or morbid consequences. However, considering the dynamic of life, more may be learned from the adaptive strategies developed during this period of high potentiality which is embryonic life, in response to such specific alterations. It should be borne in mind that these strategies may involve ``exotic'' pathways and that thorough examination of biochemical and physiological characteristics of these animals would be of potentially high significance in the understanding of the role of a given pathway.