Gary A. Klug

Exercise-induced fibre type transitions with regard to myosin, parvalbumin, and sarcoplasmic reticulum in muscles of the rat

Effects of a long-term, high intensity training program upon histochemically assessed myofibrillar actomyosin ATPase, myosin composition, peptide pattern of sarcoplasmic reticulum (SR), and parvalbumin content were analysed in muscles from the same rats which were used in a previous study (Green et al. 1983). Following 15 weeks of extreme training, an increase in type I and type IIA fibres and a decrease in type IIB fibres occurred both in plantaris and extensor digitorum longus (EDL) muscles. In the deep portion of vastus lateralis (VLD), there was a pronounced increase from 10±5% to 27±11% in type I fibres. No type I fibres were detected in the superficial portion of vastus lateralis (VLS) both in control and trained animals. An increase in slow type myosin light chains accompanied the histochemically observed fibre type transition in VLD. Changes in the peptide pattern of SR occurred both in VLS and VLD and suggested a complete transition from type IIB to IIA in VLS and from type IIA to I in VLD. A complete type IIA to I transition in the VLD was also suggested by the failure to detect parvalbumin in this muscle after 15 weeks of training. Changes in parvalbumin content and SR tended to precede the transitions in the myosin light chains. Obviously, high intensity endurance training is capable of transforming specific characteristics of muscle fibres beyond the commonly observed changes in the enzyme activity pattern of energy metabolism. The time courses of the various changes which are similar to those in chronic nerve stimulation experiments, indicate that various functional systems of the muscle fibre do not change simultaneously.

Prolonged exercise induces structural changes in SR Ca2+-ATPase of rat muscle

Sarcoplasmic reticulum (SR) isolated from the deep red portion of the gastrocnemius muscle of Sprague-Dawley rats after a single bout of prolonged exercise was shown to have depressed Ca(2+)-stimulated Mg(2+)-dependent ATPase activity over a temperature range of 15 to 42.5 degrees C when compared to SR obtained from control muscle. Inclusion of the calcium ionophore, A23187, failed to restore the depressed ATPase activity from SR of exercised muscle to control values, but it did normalize the stimulatory effect of temperature on ATPase activity. This depression was also manifested as an increased activation energy when the data were converted to an Arrhenius plot. SR vesicles from both groups showed no differences or discontinuities in plots of steady-state fluorescence anisotropy. When the binding characteristics of the fluorescent probe, fluorescein isothiocyanate (FITC), were analyzed, SR vesicles prepared from exercised muscle displayed a 40% reduction in binding capacity with no apparent change in Kd. These findings support the conclusion that a single bout of exercise induces a structural change in the Ca(2+)-ATPase protein of rat red gastrocnemius muscle that is not a direct result of gross lipid alterations or increased muscle temperature.

Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum.

Prolonged exercise decreased the rate of Ca+ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 μM AgNO3. [3H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca2+ released by Ag+ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca2+ release in the presence of ruthenium red, a known inhibitor of the Ca2+ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca2+ release from SR by directly modifying the Ca2+ release channel.

Relationships between early alterations in parvalbumins, sarcoplasmic reticulum and metabolic enzymes in chronically stimulated fast twitch muscle

The present study compares the time courses of the early changes in parvalbumin content, in the properties of the sarcoplasmic reticulum (SR) and in activity and isozyme patterns of metabolic enzymes in chronically (12 h/day) stimulated fast twitch tibialis anterior (TA) muscle of the rabbit. Under the chosen conditions of stimulation, the first significant changes appeared after 6 days. Except for the delayed reduction in pyruvate kinase, the time course of the changes were the same. After 14 days of stimulation, parvalbumin decreased to 37% and Ca2+-ATPase activity of the SR to 29% of normal values. The transformation of the SR was also reflected by a 64% decrease of the 115000-Mr Ca2+-pumping peptide and a 5-fold increase in a 30000-Mr peptide. Following an identical time course, the mitochondrial activities of citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and ketoacid-CoA transferase increased 2.9, 3.0 and 3.7-fold respectively. A similar time course was observed in the M to H-type transition of the lactate dehydrogenase isozyme. The cause of these changes is discussed at it relates to altered transcriptional and/or translational activities. It is suggested that an increase in free intracellular Ca2+ caused by increased contractile activity, which is then perpetuated by the decrease in Ca2+-binding and sequestering capacities, might be the signal for such altered synthetic activities.

Relationship between parvalbumin content and the speed of relaxation in chronically stimulated rabbit fast-twitch muscle

The time courses of changes in parvalbumin (PA) content, isometric twitch tension, and half-relaxation time (1/2RT) were studied in rabbit tibialis anterior muscle following chronic 10 Hz nerve stimulation of 1–21 days. Up to 5 days stimulation had no effect on PA content, but it induced a slight (10–15%) increase in the 1/2RT. This change occurred together with the previously observed 50% decrease in Ca2+-uptake by the SR (Leberer et al. 1987). While prolonged stimulation produced no further decrease in the Ca2+-uptake by the SR, PA content declined after 5 days of stimulation. The reduction in PA content was accompanied by a progressive lengthening of the 1/2RT. However, the increase in 1/2RT was particularly pronounced after PA had fallen below 50% of its normal value. A 90% reduction in PA coincided with a 60% increase in the 1/2RT. By this time the staircase phenomenon, normally observed in fast-twitch muscle, was completely abolished. Although the changes in PA content and 1/2RT were not linearly related, these results suggest that PA plays an important role in the relaxation process of mammalian fast-twitch muscle.

Alterations in liver mitochondrial function as a result of fasting and exhaustive exercise

The effect of exercise upon liver mitochondria structure and function was examined in fasted and fed rats, following a single run to exhaustion on a motor-driven treadmill. Exercise alone and exercise coupled with fasting both produced a significant decrease in the amount of hexokinase bound to the mitochondria, as well as reduction in the ADP/O ratio and acceptor control index measured in the presence of succinate. The mitochondria of the exercised animals, when exposed to freeze-fracture analysis while in state 3, displayed fewer deflections in the fracture plane between the inner and outer membrane than those isolated from control animals. This suggests that fewer contacts existed between the two membranes. Measurements based upon the binding of 8-anilinonaphthalene 1-sulphonate indicated that there was an increase in the net negative charge on the surface of the mitochondrial membranes of the exercised animals. All of these effects could be mimicked by incubation of mitochondria from control animals with free fatty acids. This fact, coupled with the observation that washing of the mitochondria with a solution comprising 5% (w/v) albumin could reverse all of the consequences of exercise, suggests that these alterations in mitochondrial structure and function may be the result of the increase in plasma free fatty acids that accompanies long-term exercise. Furthermore, the observation that the exercise-induced changes are dynamic and readily reversible indicates that the mitochondria were not necessarily damaged, but rather that the coupling of oxidative phosphorylation may be subject to physiological regulation.

TRAINING-INDUCED ALTERATIONS IN LACTATE DEHYDROGENASE REACTION KINETICS IN RATS: A RE-EXAMINATION

The kinetics and the isozyme composition of lactate dehydrogenase (LDH) were measured in rat plantaris muscle during a 26 week endurance‐training program. Alterations in the LDH isozyme pattern were detectable after 6 weeks as the percentage of the M4 isozyme was reduced from 89 to 76% and the total percentage of M subunits compared with H subunits declined by 8%. At 16 weeks, M4 accounted for only 62% of the total. The replacement of M with H subunits continued when training was prolonged as M4 represented only 52% of the total isozymes at 26 weeks. Conversely, training for 6 and 16 weeks produced no changes in either Vmax or Km. At 26 weeks, these values declined for both the forward (pyruvate to lactate) and backward reactions. The rate constants for both reactions were also reduced. These data suggest that changes in LDH isozyme pattern do not contribute significantly to the enhancement of lactate oxidation that may occur after training. They also suggest that the functional significance of alterations in LDH structure and/or function are best determined from analysis of the overall reaction kinetics as opposed to individual characteristics such as the isozyme pattern, Km or Vmax.

Glucose 6‐phosphate alters rat skeletal muscle contractile apparatus and sarcoplasmic reticulum function

We investigated the effects of glucose 6‐phosphate (G6P) on skeletal muscle contractile apparatus and sarcoplasmic reticulum (SR) function. Using rat extensor digitorum longus fibres, the presence of 5 mM G6P decreased the Ca2+ sensitivity of both force production and actomyosin ATPase (AM‐ATPase) activity. Conversely, maximal Ca(2+)−activated force was unaffected while maximal AM‐ATPase activity was increased by 37%. In SR vesicles isolated from rat gastrocnemius, G6P markedly altered Ca2+ handling. It increased Ca(2+)‐stimulated Ca(2+)‐ATPase activity but depressed the net rate of Ca2+ uptake. This latter effect appears to be due to G6P‐stimulated Ca2+ release. When G6P was added to Ca(2+)‐loaded vesicles, a small, transient release of Ca2+ was elicited. In addition, G6P lowered the threshold for Ca(2+)‐induced Ca2+ release but depressed the net rates of both AgNO3‐ and caffeine‐induced releases. It is possible that the accumulation of G6P during muscular activity may adversely affect muscle force production and contribute to the fatigue process via its action on the contractile apparatus and SR.

[56] Regulation of parvalbumin concentration in mammalian muscle

Publisher Summary Parvalbumin (PA), an acidic Ca 2+ - and Mg 2+ -binding protein of approximately 12,000 M r , is thought to act in skeletal muscle as a cytosolic Ca 2+ - and Mg 2+ -buffering compound. In mammalian muscle, it has been detected exclusively in fast-twitch fibers. Several studies have shown that the PA concentration in skeletal muscle is subject to regulation by exogenous factors and may be altered experimentally by changes in contractile and/or motoneuron activity. It has also been shown that PA is reduced in hereditary mammalian muscle diseases. The influence of neural activity upon PA expression is clearly shown by denervation, cross-reinnervation, and nerve-stimulation experiments. Denervation of neonatal and adult fast-twitch muscles suppresses PA synthesis. Its concentration remains low in denervated, presumptive fast-twitch muscles of the newborn rabbit. PA synthesis is suppressed in fast-twitch muscle after cross-reinnervation with a nerve that normally supplies a slow-twitch muscle. In view of the different activity patterns of fast and slow motoneurons, it seems likely that PA expression is under the positive control of phasic, high-frequency activity as delivered by fast motoneurons.

The relationship between plasma free fatty acids and liver mitochondrial function in vivo.

P/O ratio, state 3 and 4 respiration rates, and acceptor control index (ACI) were assessed in rat liver mitochondria following an overnight fast and single bout of treadmill exercise of 30-180 min. P/O was unaffected by fasting and 30 min of exercise; however, ACI was reduced because of an increase in state 4 respiration. Fasting, followed by running for 1 h or more decreased P/O approx. 40% and ACI by 50%, an effect that could be attributed to a reduction in state 3 respiration. The decrease in P/O was reversed 15 min after the cessation of exercise, whereas ACI remained depressed. All these functional alterations were mimicked by incubation of isolated mitochondria with palmitate and reversed by washing them with albumin. No direct correlation between plasma free fatty acids and the alterations in mitochondrial respiration was apparent. These data demonstrate that the decrease in the normal coupling of oxidation and phosphorylation in liver mitochondria produced by fasting/exercise is reversed rapidly in vivo. Furthermore, it is apparent that, if fatty acids act as a regulatory agent under these conditions, they do not do so solely on the basis of their plasma concentration.