Quantitative electrophoretic analysis of myosin heavy chains in single muscle fibers.

To better understand the molecular basis of the large variation in mechanical properties of different fiber types, there has been an intense effort to relate the mechanical and energetic properties measured in skinned single fibers to those of their constituent cross bridges. There is a significant technical obstacle, however, in estimating the number of cross bridges in a single fiber. In this study, we have developed a procedure for extraction and quantification of myosin heavy chains (MHCs) that permits the routine and direct measurement of the myosin content in single muscle fibers. To validate this method, we also compared MHC concentration measured in single fibers with the MHC concentration in whole fast-twitch (psoas and gracilis) and slow-twitch (soleus) muscles of rabbit. We found that the MHC concentration in intact psoas (184 microM) was larger than that in soleus (144 microM), as would be expected from their differing mitochondrial content and volume of myofibrils. We obtained excellent agreement between MHC concentration measured at the single fiber level with that measured at the whole muscle level. This not only verifies the efficacy of our procedure but also shows that the difference in concentration at the whole muscle level simply reflects the concentration differences in the constituent fiber types. This new procedure should be of considerable help in future attempts to determine kinetic differences in cross bridges from different fiber types.

Trading force for speed: why superfast crossbridge kinetics leads to superlow forces.

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only approximately 1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.

Cellular adaptations in the diaphragm in chronic obstructive pulmonary disease.

BACKGROUND: In patients with severe chronic obstructive pulmonary disease, the diaphragm undergoes physiologic adaptations characterized by an increase in energy expenditure and relative resistance to fatigue. We hypothesized that these physiologic characteristics would be associated with structural adaptations consisting of an increased proportion of less-fatigable slow-twitch muscle fibers and slow isoforms of myofibrillar proteins. METHODS: We obtained biopsy specimens of the diaphragm from 6 patients with severe chronic obstructive pulmonary disease (mean [+/-SE] forced expiratory volume in one second, 33+/-4 percent of the predicted value; residual volume, 259+/-25 percent of the predicted value) and 10 control subjects. The proportions of the various isoforms of myosin heavy chains, myosin light chains, troponin, and tropomyosin were determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. We also used immunocytochemical techniques to determine the proportions of the various types of muscle fibers. RESULTS: The diaphragm-biopsy specimens from the patients had higher percentages of slow myosin heavy chain I (64+/-3 vs. 45+/-2 percent, P<0.001), and lower percentages of fast myosin heavy chains IIa (29+/-3 vs. 39+/-2 percent, P=0.01) and IIb (8+/-1 vs. 17+/-1 percent, P<0.001) than the diaphragms of the controls. Similar differences were noted when immunohistochemical techniques were used to compare the percentages of these fiber types in the two groups. In addition, the patients had higher percentages of the slow isoforms of myosin light chains, troponins, and tropomyosin, whereas the controls had higher percentages of the fast isoforms of these proteins. CONCLUSIONS: Severe chronic obstructive pulmonary disease increases the slow-twitch characteristics of the muscle fibers in the diaphragm, an adaptation that increases resistance to fatigue.

Chronic congestive heart failure elicits adaptations of endurance exercise in diaphragmatic muscle.

BACKGROUND: During rest and exercise, patients with heart failure hyperventilate; therefore, the diaphragm can be viewed as undergoing constant moderate-intensity exercise. Accordingly, we hypothesized that heart failure elicits adaptations in the diaphragm similar to those elicited by endurance exercise in the limb muscles of normal subjects. METHODS AND RESULTS: Costal diaphragmatic biopsy samples were obtained from 7 normal subjects (age, 36 +/- 20 years) and 10 patients (age, 50 +/- 6 years; left ventricular ejection fraction, 18 +/- 8%) at the time of transplant or left ventricular assist-device placement. We measured the distribution of myosin heavy chain isoforms I, IIa, and IIb by SDS gel electrophoresis. We also measured the activities of the following enzymes: citrate synthase, a marker of oxidative metabolism; beta-hydroxyacyl-CoA dehydrogenase, a marker of lipolytic metabolism; and lactate dehydrogenase, a marker of glycolytic metabolism. In normal subjects, the distribution of myosin heavy chain isoforms I, IIa, and IIb was 43 +/- 2%, 40 +/- 2%, and 17 +/- 1%, respectively. In contrast, in heart failure subjects, the fiber distribution was 55 +/- 2%, 38 +/- 2%, and 7 +/- 2% for types I, IIa, and IIb, respectively. Therefore, in heart failure, myosin heavy chain I is increased (P < .0001) and myosin heavy chain IIb decreased from normal levels (P < .001). Additionally, citrate synthase activity (normal, 0.33 +/- 0.14; heart failure, 0.54 +/- 0.21 mumol.min-1.mg protein-1; P < .05) and beta-hydroxyacyl-CoA dehydrogenase activity (normal, 0.27 +/- 0.04; heart failure, 0.38 +/- 0.02 mumol.min-1.mg protein-1; P < .05) were greater in heart failure patients than in normal subjects, whereas lactate dehydrogenase activity was significantly less in heart failure patients than in normal subjects (normal, 11.6 +/- 4.6; heart failure,: 4.3 +/- 2.2 mumol.min-1.mg protein-1; P < .01). CONCLUSIONS: In the diaphragm in heart failure, there is a shift from fast to slow myosin heavy chain isoforms with an increase in oxidative capacity and a decrease in glycolytic capacity. These diaphragmatic muscle changes are consistent with those elicited by endurance training of the limb muscles in normal subjects.

Changes in myofibrillar protein composition of human diaphragm elicited by congestive heart failure.

We describe the changes in proportions of myofibrillar proteins elicited by chronic congestive heart failure in the costal diaphragm (DIA) of humans using one and two-dimensional electrophoretic techniques. Three myosin heavy chain (MHC) isoforms were found in the DIA from control subjects: slow MHC I (43 +/- S.E. 2%), fast MHC IIa (41 +/- 2%) and fast MHC IIb (17 +/- 1%). In heart failure DIA, the percentage of MHC I was increased to 57 +/- 2%, while that of MHC IIb was decreased to 8 +/- 2 (P < 0.001 for both cases). Similarly, this DIA had higher molar ratios (%) of the slow myosin light chain isoforms (i.e. 1sa, 1sb, and 2s), and lower molar ratios of the fast isoforms (i.e. 1f, 2f, and 3f) than control DIA. Heart failure DIA also contained lower proportions of both alpha-tropomyosin and fast isoforms of troponin-T, I and C than control DIA. These results indicate that heart failure elicits fast-to-slow transformations of both myosin and regulatory proteins of human costal DIA. These changes can be viewed as an increase in slow-twitch characteristics of the DIA and differ from the adaptations elicited by heart failure in limb muscles.

Creatine depletion elicits structural, biochemical, and physiological adaptations in rat costal diaphragm.

To elucidate adaptations elicited by creatine (Cr) depletion in the costal diaphragm (Dia), 16 12-wk-old male Fisher 344 rats had 2% beta-guanidinopropionic acid (beta-GPA), a competitive inhibitor of Cr transport into muscle, added to their food; a control group (Con) of 16 rats ate normal rat chow. After 18 wk, beta-GPA and Con Dia did not differ histochemically with respect to fiber-type distribution; however, the cross-sectional area of type II(b + x) fibers was 33% less in beta-GPA than Con Dia. Biochemically, the proportion of myosin heavy chain IIb in beta-GPA Dia was decreased 42% from Con Dia, whereas the proportions of myosin heavy chains I and IIa were increased. Physiologically, both peak twitch tension and tetanic tension in beta-GPA Dia were decreased 40% from Con. To assess fatigability, we used the protocol of Kelsen and Nochomovitz (J. Appl. Physiol. 53; 440-447, 1982) for 2-6 min duration; the percentage of initial force exhibited by beta-GPA Dia was approximately twice that of Con Dia. We conclude that these structural, biochemical, and physiological adaptations elicited by Cr depletion can all be explained by selective atrophy of IIb muscle fibers in the Dia.

[A mechanism for dual-stage kinetics of actomyosin superprecipitation]. / Mekhanizm dvustadiinoi kinetiki superpretsipitatsii aktomiozina.

It has been shown that a step-like superprecipitation of actomyosin is determined by parallel processes of two types of superprecipitation: "immediate", which is characterized by a rapid one-step increase in turbidity, and "spontaneous", when slow increase growth of turbidity follows a clear phase. It is suggested that this phenomenon is based on heterogeneous nature of particle sizes in the original suspension, which, under certain conditions, leads to "constriction" of larger particles and "clearing" of smaller ones.

Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction.

A point mutation in the heavy chain of cardiac myosin, resulting in replacement of an arginine (Arg) with glutamine (Gln), has been linked to hypertrophic cardiomyopathy in humans (Geisterfer-Lowrance, A. A. T., Kass, S., Tanigawa, G., Vosberg, H.-P., McKenna, W., Seidman, J. G., and Seidman, C. E. (1990) Cell 62, 999-1006). To determine the functional impact of this mutation, baculovirus-driven coexpression of myosin heavy and light chains has been developed. The Arg-403-->Gln mutation resulted in cardiac myosin with normal ATPase activity in the absence of actin. However, in the presence of actin, ATPase activity was greatly reduced (Vmax decreased > 3.5-fold and K(app) increased > 3-fold). In vitro motility was reduced nearly 5-fold by this single amino acid mutation. Thus, Arg-403 likely contributes to an important interaction at the actin interface of myosin. Replacement of Arg-403 with Gln leads to decreased rate(s) of transition within the actin-myosin crossbridge cycle. In humans, this mutation will result in decreased power output per unit area of cardiac muscle, likely providing a stimulus for hypertrophy.

[Interconnection between the two-stage kinetics of actomyosin Mg2+-ATPase and breaks in Arrhenius plots]. / Vzaimosviaz' mezhdu dvustadiinoi kinetikoi Mg2+-ATFazy aktomiozina i izlomami na grafikakh Arreniusa.

It has been shown that breaks in Arrhenius plots take place only when the two-stage mechanism of actomyosin ATPase is realized. Removal of two-stage behavior of ATPase leads to the strengthening of Arrhenius plots. It can be achieved by decomposition of two-stage kinetic curves into two one-stage (higher and lower temperature) constituent curves, or by "switching off" ATPase reaction over one of the two one-stage mechanisms by changing KC1 or ATP concentrations. In both cases Arrhenius plots are linear and Ea values, as well as maximal ATPase activity for higher temperature mechanism are twice as much as those for the lower temperature mechanism. On the contrary, non-linear Arrhenius plots show the lowering of Ea with an increase of temperature. We conclude that the breaks in Arrhenius plots can be due to the shift of equilibrium between two one-stage mechanisms of actomyosin ATPase, whose simultaneous course leads to the two-stage kinetics of this reaction.

[The character of actin-myosin interaction at two stages of the superprecipitation reaction]. / Kharakter aktin-miozinovogo vzaimodeistviia na dvukh stadiiakh reaktsii superpretsipitatsii.

It has been shown that two-stage kinetics of superprecipitation (SPP) and ATPase of natural and synthetic actomyosin can be modulated by changing Mg-ATp2- concentration. The I stage is activated at low substrate concentrations, and the II stage--at high concentrations. Resynthesis of ATP completely inhibited the II stage of SPP (and ATPase) and produced no effect in the clearing phase, as well as in the I stage of these reactions. We conclude that active myosin bridges function during the I stage of SPP. However, the II stage ends with the formation of rigorous bridges. It is suggested that division of two different types of actomyosin complexes which participated in the alternative kinetic mechanisms of both, SPP and ATPase reactions, takes place at the moment when ATP is bound in active sites of myosin and dependent on substrate concentration.

[The comparative characteristics of myofibrillar protein transformation in muscles of different phenotypes]. / Sravnitel'naia kharakteristika belkovoi transformatsii miofibrill myshts raznogo fenotipa.

Studies have been made on Mg2(+)-ATPase, Ca2(+)-sensitivity, superprecipitation and fractional composition of natural and desensitized actomyosin from myocardium, slow and fast skeletal muscles after physical training (swimming, gravitational loading) and after monthly readaptation. Physical overloading makes physicochemical properties and protein composition of actomyosin from the myocardium and slow skeletal muscles similar to those in fast skeletal ones. Changes in actomyosin from the myocardium and slow skeletal muscles are more profound, whereas the recovery of the initial properties during readaptation reveals high plasticity of muscles of these phenotypes. Changes in Ca regulation depend mainly on muscle phenotype. Different plasticity of muscles of various phenotypes during readaptation results from differences in the synthesis of protein components of myofibrils.

[pH-dependent changes in the two-stage kinetics of superprecipitation reaction of natural actomyosin]. / pH-zavisimye izmeneniia dvustadiinoi kinetiki reaktsii superpretsipitatsii natural'nogo aktomiozina.

The effect of pH on the two-stage kinetics of the superprecipitation (SPP) reaction of natural actomyosin was investigated. It was shown that the experimental dependencies appear as two intersecting bell-shaped curves reflecting the effects of pH on individual steps of the SPP reaction which are mediated by different molecular mechanisms. It was supposed that the both reaction mechanisms involve actomyosin complexes which have different structural states and differ also by the degree of dissociation in the presence of ATP. The shifts in the dynamic equilibrium between the two states of actomyosin may induce pH-modulations in the two-stage kinetics of SPP and, presumably, ATPase.

[The effect of magnesium ions on the two-stage kinetics of superprecipitation and ATPase activity of natural actomyosin]. / Deistvie ionov magniia na dvustadiinuiu kinetiku superpretsipitatsii i ATPazy natural'nogo aktomiozina.

The effect of magnesium ions on the two-stage kinetics of superprecipitation (SPP) and ATP activity of natural skeletal muscle actomyosin was studied. It was found that the changes in the ratios of two independent steps of SPP and ATPase activity are mainly induced by the Mg-ATP2- complex, but not by free Mg2+. These changes in the kinetics of SPP and ATPase are regarded as being due to the shift in the dynamic equilibrium between the two types of the actomyosin complexes in solution, each of which is characterized by different reaction mechanisms. The role of the Mg-ATP2(-)-induced alteration of at least two structural-and-functional states of actomyosin in muscle contractibility is discussed.

[A possible role of myosin conformers in provision of dual-stage superprecipitation kinetics of natural actomyosin]. / Vozmozhnaia rol' miozinovykh konformerov v obespechenii dvustadiinoi kinetiki superpretsipitatsii natural'nogo aktomiozina.

It has been shown that synchronous starting and successive accomplishment of superprecipitation on the two types of actomyosin complexes lead to the two-stage kinetics of this reaction. By means of a temperature change different balance of two types of actomyosin macromolecules can be achieved. We conclude that two different structural forms (conformers) of myosin cause two non-equivalent functional states of the whole actomyosin complex.

[Participation of two types of actomyosin complexes in the process of superprecipitation]. / Uchastie dvukh tipov aktomiozinovykh kompleksov v protsesse superpretsipitatsii.

It has been shown that addition of turbidity changes accompanying two different types of actomyosin complexes synchronous function in solution allows observation of biphasic kinetics of superprecipitation.

[Effect of cardioactive compounds on myocardial actomyosin in the centrifuged rat]. / Vliianie kardioaktivnykh soedinenii na aktomiozin miokarda krys posle deistviia peregruzok.

It has been shown that during centrifugation the modulating effect of cardioactive compounds, particularly adrenalin and obsidan, varies in similarity to that of Ca ions. The reactivity of the native actomyosin complex of the heart of white rats to such agents declines during centrifugation. This may be associated with changes in regulatory protein components through which the modulating effect of the above compounds is mediated. Differences in the reactivity to adrenalin and obsidan that persist after 2-month readaptation can be attributed to the heterogeneous recovery of properties of individual subunits of regulatory proteins.

[Changes in the physico-chemical properties of contractile and regulatory proteins in muscles of different types during overload and readaptation]. / Izmenenie fiziko-khimicheskikh svoistv sokratitel'nykh i reguliatornykh belkov raznykh tipov myshts pri peregruzke i v khode readaptatsii.

After 15-day exposure to +5 Gx the rate of superprecipitation, Mg2+-ATPase activity and actomyosin ATPase of slow muscles (m. soleus and medial head of m. triceps brachii) of white rats increased greatly. In actomyosin of fast muscles (m. brachialis and m. extensor digitorum longus) the exposure induced weaker and opposite changes in the superprecipitation rate and Mg2+-ATPase activity. The changes in actomyosin of the fast muscles were associated with shifts only in regulatory components while those of the slow muscles were produced by shifts in contractile proteins as well. This provided for a better recovery of the initial value of the superprecipitation rate and Mg2+-ATPase activity of actomyosin of the fast muscles a month after the exposure.