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(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) ( 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 ( 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 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):
K
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 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
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]
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
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 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 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 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 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.
Volume 270,
Number 34,
Issue of August 25, pp. 19914-19920, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
I. ALTERATIONS IN MYOFIBRILLAR FUNCTION (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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)).
-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).
Mouse Model of Muscle Creatine Kinase-deficient Mice
Mice bearing a null mutation of the M-CK gene were obtained
as described previously (van Deursen et al., 1993).
Heterozygous mutants were interbred to generate mice deficient in M-CK.
Offsprings were genotyped 2 weeks after birth., 25; KH
PO
, 1.2; and
MgSO, 1.2.HPO, 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.
Mechanical Experiments
Muscle Preparation
Muscle fiber bundles were
dissected from soleus or gastrocnemius or from papillary muscles of the
left ventricle of mice in a zero-Ca Krebs solution,
pH 7.4. Bundles were incubated for 1 h in a relaxing solution (pCa 9, see solutions below) containing 1% Triton X-100 to
solubilize the membranes and were then transferred to the relaxing
solution without detergent and kept at 4 °C until use. After the
skinning procedure, one bundle was snared at both ends with hair
emerging from stainless steel tubes in the experimental apparatus. It
was adjusted to slack length, stretched by 20%, and subjected to an
activation/relaxation cycle. Sarcomere length was controlled by laser
diffraction (10-milliwatt He-Ne laser, Spectra-Physics, Inc., Mountain
View, CA). The length and diameter of the muscles were measured by use
of a graticule in the dissecting microscope. Muscles were immersed in
2.5-ml chambers arranged around a disk in a temperature-controlled bath
positioned on a magnetic stirrer. Each solution was well stirred at
high speed. All experiments were performed at 22 °C.
Experimental Apparatus
The tubes were connected to
a transducer (model AE 801, SensoNor Microelectroniks, Horten, Norway)
and a vibrator as described previously (Mayoux et al., 1994).
The bandwidth of the transducer and tube was 2 kHz. The permanent
magnet and coil came from a standard loudspeaker (Pioneer TS-130A,
Pioneer Electric and Research Corp., Forest Park, IL). The coil was
glued to a glass tube axis (2 mm in diameter) driven in an axial ball
bearing (total moving mass < 1.5 g). A flag with a narrow window was
glued on the glass axis between a lamp and a position detector (type
S1543, Hamamatsu, Japan), allowing measurements of the displacement
length. A feedback with the length signal combined with a power
amplifier allowed control of muscle length. The system had a rise time
of about 1 ms without overshoot. Length and tension changes were
monitored on a digital storage oscilloscope (OS4020, Gould, Inc.,
Cleveland, OH). Tension tracings were digitized at 20 kHz (12-bit
analog/digital converter), analyzed on-line using a PC compatible
computer, and stored on a videotape.Solutions
Solutions were calculated using the
computer program of Fabiato(1988). All solutions were calculated to
contain (mM): EGTA, 10; imidazole, 30; Na,
30.6; Mg
, 3.16; and dithiothreitol, 0.3; ionic
strength was adjusted to 0.16 M with potassium acetate. pH was
adjusted to pH 7.1 with acetic acid. In relaxing and rigor solutions, pCa was 9. In activating solution, pCa was 4.5.
Relaxing and activating solutions also contained 3.16 mM MgATP
and 12 mM PCr. Rigor solutions were obtained by mixing two
solutions of pMgATP 2.5 and 6 or pMgATP 4 and 6. EGTA
was obtained from Sigma. PCr (Neoton, Schiapparelli Farmaceutica
SEARLE, Turin, Italy) was a kind gift of Prof. E. Strumia.
Experimental Protocols
pCa/Tension Relations
pCa/tension
relations were determined under isometric conditions by briefly placing
each fiber into solutions of increasing calcium concentration until
maximal tension was reached. Data were fitted using linearization of
the Hill equation for relative tensions above 10% and under 90% as
follows: T =
[Ca]
/(K + [Ca
]
) were T is relative tension, K a constant, and n the Hill coefficient. n and pCa for half-maximal
activation (pCa
=
-log
K/n) were calculated for each
fiber by means of linear regression analysis. Resting tension was
measured at pCa 9. Active tension (expressed as
mN/mm
) was the total tension at pCa 4.5
minus resting tension.pMgATP/Rigor Tension
Relations
pMgATP/rigor tension relations were
established by stepwise decreasing ATP concentrations until maximal
rigor tension was obtained. The fiber was then placed in the relaxing
solution for 10 to 15 min before a new set of rigor solutions was
applied. Data were fitted using the Hill equation. The pMgATP
for half-maximal rigor tension, pMgATP =
(-log
K)/n, was calculated for each
experimental condition using linear regression analysis.
Stiffness and Kinetic Measurements
To determine
fiber bundle stiffness and the rate constant of tension recovery in
cardiac preparations, quick length changes (0.3-3% of initial
muscle length) were applied in the relaxing and activating solutions.
Twelve successive stretches and releases were made, starting with the
relaxing solution. Only responses to stretches were used for
calculations. Each value for each fiber was the mean of five to seven
stretches of varying amplitudes performed in a given experimental
condition. The spike of tension in phase with the length change
characterized the elastic phase (Huxley and Simmons, 1971). Stiffness
was the extreme tension reached during stretching
(mN/mm) divided by the length change (µm). A
first series of length changes was imposed in the relaxing solution to
assess passive properties of each fiber. Resting stiffness was
calculated by linear regression analysis on the responses to stretches.
Then a second series of length changes was initiated in control
activating solution. The tension level before the first stretch was
taken as the maximal tension and used for normalizations. Active or
rigor stiffness were calculated as the difference between total
stiffness minus resting stiffness. The rate constant of tension
recovery after quick stretches was calculated by a least square
regression analysis, according to a single exponential model, between
50% and 80% of recovery and using stretches of more than 1%.Enzyme Analysis
Enzyme activities were determined spectrophotometrically at
340 nm (Gilford Spectrophotometer, Corning, NY), 30 °C, by using
coupled enzyme systems. Results are given in IU/g wet weight. Myokinase
activity was determined at pH 7.1 using the coupled enzyme assay of
hexokinase and glucose-6-phosphate dehydrogenase producing NADPH.
Activity was assayed in a medium containing (in mM): HEPES,
20; MgCl, 5; dithiothreitol, 0.5; ADP, 1.2; glucose, 20;
NADP, 0.6; and 2 IU/ml glucose-6-phosphate dehydrogenase and
hexokinase. Glyceraldehyde-3-phosphate dehydrogenase was determined at
pH 7.6, in a medium containing (in mM): triethanolamine, 82.5;
3-phosphoglycerate, 6; ATP, 1.1; EDTA, 0.9; MgSO, 1.7;
NADH, 0.2; and 15 IU/ml 3-phosphoglycerate kinase. Pyruvate kinase
activity was measured at pH 7.6 in a medium containing (in
mM): triethanolamine, 82.5; phosphoenolpyruvate, 0.54;
MgSO, 2.5; KCl, 10; ADP, 4.7; NADH, 0.2; and 9.2 IU/ml
lactate dehydrogenase. Fructose-6-phosphokinase was determined at pH
8.5 in a medium containing (in mM): Tris-HCl, 70;
MgSO, 1.4; KCl, 4.5; phosphoenolpyruvate, 0.71; fructose
1,6-diphosphate, 0.64; fructose 6-phosphate, 1.8; ATP, 1.1; NADH, 0.2;
and 9.6 IU/ml lactate dehydrogenase and 4.2 IU/ml pyruvate kinase.
Lactate dehydrogenase activity was determined at pH 7.4 using a medium
containing (in mM): KHPO, 20; KCl,
120; dithiothreitol, 0.5; pyruvate, 10; NADH, 0.2.Myosin Isoforms
Myosin Preparation
Frozen muscles were thawed on
ice, cut into small pieces, and washed with 5 volumes of 20 mM NaCl, 5 mM sodium phosphate, and 1 mM EGTA (pH
6.5). Myosin was then extracted with 3 volumes of 100 mM sodium pyrophosphate, 5 mM EGTA, and 1 mM dithiothreitol (pH 8.5); after 30 min of gentle shaking, the
mixture was centrifuged at 10,000 g. The supernatant
containing myosin was diluted with 1 volume of glycerol and stored at
-20 °C (d'Albis et al., 1979).
Electrophoresis of Native Myosin Isoforms
Gel
running buffer consisted of 20 mM sodium pyrophosphate (pH
8.5), 10% glycerol, 0.01% 2-mercaptoethanol, and 2 mM MgCl. Cylindrical (6 0.5 cm) gels contained 4%
polyacrylamide (3.88% acrylamide and 0.12% N,N`-methylenebisacrylamide). Between 1 and 5 µg of myosin
was loaded on each gel. Electrophoresis was carried out at a constant
voltage of 90 V, for 22 h, between 0 and 2 °C (d'Albis and
Gratzer, 1973; Hoh, 1975).
Electrophoresis of Myosin Heavy Chains and
Quantification
Electrophoresis was performed as described by
Talmadge and Roy(1993). Minigels were used in the Bio-Rad Mini-PROTEAN
II Dual Slab Cell. Electrophoresis took place in a cold cupboard, at 10
°C for 28 h. The gels were stained with Coomassie Blue R-250, and
the relative amounts of the different myosin heavy chains were measured
using a densitometer equipped with an integrator.Statistical Analysis
Values were expressed as mean ± S.E. Student's t test was used to compare the means between control and
transgenic animals or inside groups between two experimental
conditions. Statistical significance was reached when p <
0.05.
Anatomical Data
Comparing control and transgenic
mice, no statistical difference was observed between heart, lung,
liver, kidney, and body weights (Table 1). The heart weight to
body weight ratio was in consequence not altered in transgenic animals.
Thus, no sign of organ dysfunction or change in muscle mass or cardiac
insufficiency could be detected.
Tension and Calcium Sensitivity of Skinned
Fibers
Fibers of similar diameter were dissected from control
and transgenic mice. Diameters were, respectively, for control and
transgenic: 207 ± 23 and 179 ± 16 µm for ventricle,
160 ± 15 and 138 ± 20 µm for soleus, 155 ± 26
and 170 ± 26 µm for gastrocnemius. No statistical difference
was observed, which allowed accurate comparison of mechanical
performances. Table 2shows mechanical parameters of skinned
fibers, normalized per cross-sectional area, in resting (pCa
9) and activating (pCa 4.5) conditions. Whatever the muscle,
resting tensions were not modified in transgenic animals. Although
clear differences existed between active forces developed by the
different muscles, no differences were observed between control and
transgenic animals.
) 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.
. 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.
Effect of Phosphocreatine on Relaxation of Rigor
Tension
The next series of experiments was undertaken to study
the influence of PCr on the relaxation of rigor tension in control and
transgenic muscles. Rigor tension is the tension induced in the virtual
absence of calcium by decreasing MgATP concentration. We have shown in
ventricular muscle that the development of rigor tension is greatly
influenced by CK bound in myofibrils. The concentration of MgATP
necessary to obtain half-maximal rigor is greatly reduced when PCr is
provided as a substrate. In order to characterize more precisely this
effect in control and transgenic mice, complete pMgATP/rigor
tension relations have been established in presence or absence of PCr
in sets of solutions of decreasing MgATP concentrations. In Fig. 2, pMgATP/rigor tension relations have been drawn
using Hill equation and pMgATP (pMgATP
for half-maximal rigor force) values given in Table 2for
ventricle, soleus, or gastrocnemius. When PCr was added, a clear shift
of the relation was observed in control muscles. While pMgATP/tension relations without PCr were identical in control
and transgenic animals, no shift could be evidenced in the presence of
PCr for the three muscle types of transgenic animals. pMgATP
values in the presence of PCr, as well as
CK efficacy, which is defined as the difference between pMgATP
values in the presence and absence of PCr,
were both highly significantly different between control and transgenic
mice (Table 2). This result is in complete agreement with the
absence of M-CK in these animals and clearly shows that myofibrils from
transgenic mice have no enzyme able to utilize PCr.
), 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.
Responses of Ventricular Skinned Fibers to Quick Length
Changes
Active stiffness as measured by the tension responses to
a quick length change was not significantly different in ventricular
fiber bundles of control and transgenic mice (450 ± 57
mN/µm/mm (n = 8) versus 644 ± 69 mN/µm/mm (n = 6) respectively). It should be noted, however, that a
small tendency toward an increase in both tension and stiffness could
be observed, although not reaching significance, possibly as a result
of poor estimation of the fibers' effective cross-section.
However, the ratio of force to stiffness was not altered (Fig. 3) in transgenic mice showing that force developed by each
cross-bridge was preserved. When a length perturbation was applied to
activated muscle, force first increased in phase with the increase in
length and then decreased toward the value of the tension before the
length change. The rate constant of tension recovery reflects the
cross-bridge cycling rate, while the extent of recovery characterizes
the cross-bridge state. The rate constant of tension recovery following
stretches was greatly decreased in transgenic mice compared to control
while the extent of recovery was not different (Fig. 3). This
result suggests a decrease in the cross-bridge cycling rate in
transgenic ventricular fibers.
to 38 ± 4 s
(n = 4, p < 0.05).
Isomyosin Patterns
A decrease in cross-bridge
cycling rate may arise from an altered pattern in myosin isoenzyme
distribution. In order to see if transgenic animals exhibit a change in
myosin isoforms, the myosin phenotype was determined in the different
muscles.
Glycolytic Enzymes and Myokinase
Determinations
Since no change in isomyosin pattern could be
detected in muscles of transgenic mice, the question arose as to how
MgATP and MgADP concentrations could be controlled in transgenic
animals. Indeed, it has been shown that myofibrillar CK, by keeping
high ATP/ADP ratio and low proton concentrations close to myosin
ATPase, ensures optimal efficiency of myosin ATPase in skeletal
(Bessman et al., 1980) as well as cardiac muscle
(Ventura-Clapier et al., 1987a, 1987b). Other MgATP
regenerating enzymes may exist in cytosol or may be loosely bound to
myofibrils. To check for possible overexpression of such enzymes,
glycolytic enzymes as well as myokinase activities were measured in the
different muscles. Results are shown in Table 3. Myokinase
activity was not significantly increased in transgenic animals while
some glycolytic enzymes like pyruvate kinase and
glyceraldehyde-3-phosphate dehydrogenase increased in ventricles and
glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase
increased in gastrocnemius. No significant changes were seen for soleus
muscle.
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)).
-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.
-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. 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)).
)-GPA,
-guanidinopropionic acid; PCr, phosphocreatine.
We acknowledge Patrick Lechêne
for engineering assistance, Frank Oerlemans and Chantal Janmot for
technical assistance, Valdur Saks for helpful discussions and Rodolphe
Fischmeister for continuous support.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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