Stanislav V. Kremnyov
Z. A. Podlubnaya
Institute of Theoretical and Experimental Biophysics RAS, Pushchino State University, Pushchino, Moscow Region, 142290, Russia
E-mail: podlubnaya@iteb.ru
Smooth muscle cells use actomyosin contractile apparatus in order to produce force for various physiological functions including the regulation of blood pressure and intestinal peristalsis. Organization of contractile appara- tus of smooth muscle is similar to the organization of cross-striated skeletal and cardiac muscles. Recently in smooth muscles was found new protein smitin (from words “smooth” and “titin”) with molecular weight ~1000 кDа. Molecular morphology of this protein, its localization in contractile apparatus, capability to interact with actin and myosin are similar to those for titin of skeletal and cardiac muscles. Like titin, smitin is long fibrillar molecule [1, 2]. However, we know too little about this protein so far.
Earlier we discovered amyloids properties of proteins of titin family (titin, Х-, С-, Н-proteins) in skeletal and cardiac muscles, studied polymor- phism of amyloids of these proteins, dynamics of their formation, toxicity of different amyloid aggregates and also tested the effectiveness of number of antiamyloidogenic preparations for development of therapy amyloidoses. We carried out all investigations of titin family proteins in comparison with brain amyloid Abeta peptides and discovered similar approaches to the destruction of their amyloids [3-28]. We assumed that the analog of titin discovered in smooth muscles will be able to form amyloids in vitro and to act the important part in pathogenesis of amyloidoses in smooth muscles. Standard testing of amyloids is usually carried out by the use of specific dyes Congo-red and thioflavine-T. Our preliminary tests with thioflavine-T showed the increase of intensity of dye fluorescence in the presence of smitin, which occurs usually in the presence of amyloids formed by proteins of titin family. Our further experiments will be directed on the investigation of conditions and dynamics of formation amyloids by smitin in model systems and also on the search for effective preparations disrupting its amyloids or preventing their formation.
This work was supported by Program of Presidium RAS “Fundamen-
tal sciences - for medicine”, 2010 and by grants RFBR № № 09-04-01161, 10-04-00141.
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CHANGES IN ISOFORM COMPOSITION OF LIGHT CHAINS OF SKELETAL AND CARDIAC MYOSINS UPON HIBERNATION
AND IN CARDIAC DISEASES AND MYODYSTROPHIES Z.A. Podlubnaya 1, 2, S.N. Udaltsov 1, 3
1 Institute of Theoretical and Experimental Biophysics RAS, Pushchino, Russia
2 Pushchino State University, Pushchino, Russia
3 Institute of Physicochemical and Biological Problems in Soil Science RAS, Pushchino, Russia
E-mail: podlubnaya@iteb.ru
Myosin is the main protein of different types of muscles. The mole- cule of skeletal muscle myosin consists of two heavy chains (HC) and two light chains (LC). Skeletal muscle myosin contains the set of LC and HC typical for fast and slow fibers. Cardiac muscle myosin consists of HC and LC characteristic for atrium (aHC, aLC) and for ventricle (vHC, vLC). Our investigations of the contribution of myosin LC and HC to structural, enzy- matic and regulatory properties of myosin in norm were begun in the 1980’s [1-9]. Here we are describing the results of our studies on the changes in isoform composition of myosin LC and HC of skeletal and cardiac muscles
upon adaptation of ground squirrels (Сitellus undulatus) to hibernation [10- 20] and in several pathologies [21-33].
Hibernation of mammals is evolutionary determined ability of warm- blooded animal to adapt to unfavorable conditions by the inhibition of the activity of all physiological systems including muscles with maintenance of the control over the coordination of their action. Arousal from hibernation is very rapid process. In not more than 3h the animal passes to an active state with no pathological after-effects, using only its own resources. Therefore, hibernating animals are unique objects for study of the mobilization mecha- nisms of the reserve potentialities of the organism to survive in extreme and pathological situations. The elucidation of these mechanisms may make a valuable contribution to the solution of urgent task of medicine, cosmonaut- ics, cryo-preservation of genetic resources, etc. We have revealed the differ- ences in the following properties of myosins isolated from skeletal muscles of ground squirrels at the different stages of hibernation (Mhib, Mact, Mar).
The ATPase activity of Mhib and Mar in the presence of rabbit skeletal actin was less by 60% and 20%, correspondingly, than that of Mact. The Ca- sensitivity of AMhib and AMar (in the absence of TM and TP) was less by 60% than that of Mact. Moreover, the amount of LC3 in AMhib and AMar was less by 60% and 30%, correspondingly, than that of LC3 in Mact. The data obtained point to the essential changes in myosin isoforms composition upon hibernation and arousal and to the significant contribution of the main contractile protein myosin and its LCs to the inhibition of the contractile capacity of skeletal muscles during hibernation and its restoration upon arousal. It should be also noted that in skeletal muscles of hibernating ground squirrels, a decreased quantity of “fast” myosin LC isoforms and an increased quantity of “slow” myosin LC isoforms were found. Similar changes were also observed in HC isoform composition. Our studies on the changes in isoform composition of myosin LC and HC of skeletal muscles demonstrated that these changes are reversible on arousal of animal. They correspond to the adaptive strategy of hibernation: to preserve or increase the quantity of protein isoforms characteristic for “slow” muscle fibers, as most enduring and energetically most favorable for survival under the ex- treme conditions of hibernation and for the arousal without any pathological consequences. The changes in isoform composition of muscle proteins in different heart sections of hibernating and awaking ground squirrels are also evidence for a trend of these changes to suppress (entrance into hibernation) or to recover (arousal from hibernation) normal myocardial work. In the atrium of a hibernating ground squirrel, an increased quantity of myosin LC1 of ventricular type (vLC1) was revealed (up to 60%) whereas during arousal the atrial type of myosin LC (aLC1) appears in the heart ventricle (up to 30%). It is quite appropriately because aLC1 raises the ATPase activ- ity of ventricle myosin whereas vLC1 decreases the ATPase activity of atrial
myosin. Since myosin of hibernating animals does not have unique structure and properties therefore the study on the changes of its isoform in the proc- ess of season cycle can clarify functional importance analogical events in the development of cardiac diseases of man.
We have studied the changes in light chains of cardiac myosin upon dilated cardiomyopathy (DCM). In all experiments the hearts explanted from patients after orthotopic transplantation were used. It has been shown that in the absence of marked quantitative changes of myosin HC, the com- position and the ratio of LCs changed considerably: the amounts of vLC2 decreased by 70%. At the I-II stages of DCM (NYHA’s classification) the decrease in the vLC1 amount was accompanied by the appearance of ~30%
aLC1 in ventricular myosin which not typical of it in norm. In this case the total amount of LC1 in ventricular myosin remained constant. The appear- ance of myosin aLC1 in the left heart ventricle at the early stages DCM is an evidence for the functional importance of these replacements which are not observed at the terminal stage of disease development. Our studies on AT- Pase activity of hybrid cardiac myosins confirmed this supposition. ATPase activity of hybrid myosins with vHC increased in direct dependence from the content of incorporated aLC1. It should be noted that aLC1, not typical for normal adult human ventricles, have been shown to be expressed in em- bryonic heart. They disappear at birth of animal. Taken together our data assume that the appearance of embryonic protein isoforms in failed heart is compensatory control rather than functional failure. However, in the III-IV stages of DCM we have not revealed aLC1 in ventricular myosin. In this case the changes in LCs composition are accompanied by considerable de- crease of the enzymatic activity of myopathic myosin and also of sizes and stability of its filaments. It is likely that aLC1 appearance in ventricular muscle is the result of adaptive processes which stop in the end-stage of the disease pointing to its irreversible character.
In Duchenne's dystrophy and on atrophy caused by gravitational unloading, we observed a shift of the myosin phenotype toward the increase of “fast” myosin LC and myosin HC
According to our data, in the atrium of the patients with mitral-aortal valvular diseases and myocardial ischemia, appear myosin vLC1, thus indicat- ing the terminal stages of pathological processes in the myocardium. By the use of ELISA method we studied the changes in concentration of autoantibod- ies to cardiac myosin light chains (LC1 and LC2) in the blood serum of pa- tients who have undergone the operation on heart under conditions of artificial hypothermal circulation. Concentration of the autoantibodies was measured after 3, 6, 9, 18, 24, 36 h following the reperfusion of myocardium. A high correlation was established between the duration of cross-clamping of aorta, of the duration of illness and the concentration of autoantibodies.
Thus, the comparison of reversible changes in myosin isoform com-
position upon mammal hibernation with the changes in pathology can be useful for diagnostics, prognostics and choice of approaches to the effective therapy of some socially significant diseases.
Acknowledgements. The authors thank the coworkers of citied pub- lications for the above investigations for many years.
This work was supported by grants of the President of Russian Fed- eration “Leading scientific schools”, № НШ-4981. 2006.4, НШ-217.2008.4, by Program of the Presidium RAS "Fundamental sciences for medicine", 2004-2010, by grant of RFBR № 10-04-00141, and also by the Federal Tar- get program “Scientific and scientific-pedagogic personnel of innovative Russia“, State contract № 02.740.11.0301 and State contract N 02.740.11.0710.
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DOES AN ELECTRO NEUTRAL K+/CL- ANTIPORT OCCUR IN CARDIOMYOCYTE?