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Seasonal changes in proteolytic activity and content of calpains in striated muscles of the longtailed ground squirrel Spermophilus undulatus were studied by casein zymography and Western blotting analysis. The results testify to hyperactivation of calpain proteases in the skeletal muscles of awakened animals during the "winter" activity. The observed changes are discussed in the context of adaptation of skeletal muscles of long-tailed ground squirrels to hibernation.

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Enzymatic activity of Ca2+-dependent calpain proteases as well as the content and gene expression of µ-calpain (activated by micromolar calcium ion concentrations), calpastatin (inhibitor of calpains), and titin (substrate for calpains) were investigated in cardiac muscles of rats subjected to chronic alcoholization for 3 and 6 months. There was no increase in the "heart weight/body weight" parameter indicating development of heart hypertrophy in the alcoholized rats, while a decreasing trend was observed for this parameter in the rats after 6-month modeling of alcoholic cardiomyopathy, which indicated development of atrophic changes in the myocardium. Fluorometric measurements conducted using the Calpain Activity Assay Kit did not reveal any changes in total calpain activity in protein extracts of cardiac muscles of the rats alcoholized for 3 and 6 months. Western blot analysis did not show reliable changes in the contents of µ-calpain and calpastatin, and SDS-PAGE did not reveal any decrease in the titin content in the myocardium of rats after the chronic alcohol intoxication. Autolysis of µ-calpain was also not verified, which could indicate that proteolytic activity of this enzyme in myocardium of chronically alcoholized rats is not enhanced. Using Pro-Q Diamond staining, changes in phosphorylation level of titin were not detected in cardiac muscle of rats after chronic alcoholization during three and six months. A decrease in µ-calpain and calpastatin mRNA content (~1.3-fold, p ≤ 0.01 and ~1.9-fold, p ≤ 0.01, respectively) in the myocardium of rats alcoholized for 3 months and decrease in calpastatin mRNA (~1.4-fold, p ≤ 0.01) in animals alcoholized for 6 months was demonstrated using real-time PCR. These results indicate negative effect of chronic alcohol intoxication on expression of the abovementioned genes.

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From our earlier experiments on the study of changes in titin content and the level of its phosphorylation in skeletal muscles, atrophied during space flight, hibernation, and also because of the development of alcohol-induced lesions it has been suggested that an increase in the degree of titin phosphorylation results in increased proteolytic degradation of this protein, that contributes to the development of skeletal muscle atrophy.

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Seasonal changes in the isoform composition of thick and thin filament proteins (titin, myosin heavy chains (MyHCs), nebulin), as well as in the phosphorylation level of titin in striated muscles of brown bear (Ursus arctos) and hibernating Himalayan black bear (Ursus thibetanus ussuricus) were studied. We found that the changes that lead to skeletal muscle atrophy in bears during hibernation are not accompanied by a decrease in the content of nebulin and intact titin-1 (T1) isoforms. However, a decrease (2.1-3.4-fold) in the content of T2 fragments of titin was observed in bear skeletal muscles (m. gastrocnemius, m. longissimus dorsi, m. biceps) during hibernation. The content of the stiffer N2B titin isoform was observed to increase relative to the content of its more compliant N2BA isoform in the left ventricles of hibernating bears. At the same time, in spite of the absence of decrease in the total content of T1 in the myocardium of hibernating brown bear, the content of T2 fragments decreased ~1.6-fold. The level of titin phosphorylation only slightly increased in the cardiac muscle of hibernating brown bear. In the skeletal muscles of brown bear, the level of titin phosphorylation did not vary between seasons. However, changes in the composition of MyHCs aimed at increasing the content of slow (I) and decreasing the content of fast (IIa) isoforms of this protein during hibernation of brown bear were detected. Content of MyHCs I and IIa in the skeletal muscles of hibernating Himalayan black bear corresponded to that in the skeletal muscles of hibernating brown bear.

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Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655-658). Small pieces of cardiac muscle were incubated for 2-3 weeks at 4°C in Ca²âº-depleting solution before their homogenization to decrease activity of Ca²âº-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119-123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²âº-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ~5-20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ~30-50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²âº-dependent proteases.

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In this review our data on the comparative study of amyloid properties of titin family proteins and brain Abeta-peptides are represented. Approaches to the destruction of amyloid fibrils of muscle X-protein and brain Abeta(1-42)-peptides by various chemical compounds are also described.

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This review summarizes results of our studies on titin isoform composition in vertebrate striated muscles under normal conditions, during hibernation, real and simulated microgravity, and under pathological conditions (stiff-person syndrome, post-apoplectic spasticity, dilated cardiomyopathy, cardiac hypertrophy). Experimental evidence for the existence in mammalian striated muscles of higher molecular weight isoforms of titin (NT-isoforms) in addition to the known N2A-, N2BA-, and N2B-titin isoforms was obtained. Comparative studies of changes in titin isoform composition and structure-functional properties of human and animal striated muscles during adaptive and pathological processes led to a conclusion about the key role of NT-isoforms of titin in maintenance of sarcomere structure and contractile function of these muscles.

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Changes in isoform composition, secondary structure, and titin phosphorylation in Mongolian gerbil (Meriones unguiculatus) cardiac muscle were studied after 12-day-long space flight onboard the Russian spacecraft Foton-M3. The effect of titin on the actin-activated myosin ATPase activity at pCa 7.5 and 4.6 was also studied. Almost twofold increase in titin long N2BA isoform content relative to that of short N2B isoform was found on electrophoregrams of cardiac muscle left ventricle of the flight group gerbils. Differences in secondary structure of titin isolated from cardiac muscle of control and flight groups of gerbils were found. An increase in phosphorylation (1.30-1.35-fold) of titin of cardiac muscle of the flight group gerbils was found. A decrease in activating effect of titin of cardiac muscle of the flight group gerbils on actomyosin ATPase activity in vitro was also found. The observed changes are discussed in the context of M. unguiculatus cardiac muscle adaptation to conditions of weightlessness.

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It has been revealed for the first time that sodium fullerenolate Na(4)[C(60)(OH)(∼30)] (NaFL), a water soluble polyhydroxylated [60]fullerene derivative, destroys amyloid fibrils of the Aß(1-42) peptide in the brain and prevents their formation in in vitro experiments. The cytotoxicity of NaFL was found to be negligibly low with respect to nine different culture cell lines. At the same time, NaFL showed a very low acute toxicity in vivo. The maximal tolerable dose (MTD) and LD50 for NaFL correspond to 1000 mg kg(-1) and 1800 mg kg(-1), respectively, as revealed by in vivo tests in mice using intraperitoneal drug injection. The observed pronounced anti-amyloid activity and low toxicity of NaFL make it a very promising lead drug for the development of potent fullerene-based therapeutic approaches for the treatment of amyloidoses, such as Alzheimer's disease and others.

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It has been shown for the first time by transmission electron microscopy that the hydrated fullerene C60 inhibited the fibrillization of amyloid-beta25-35 peptide. The fullerene affected the amyloid-beta25-35 assembly, manifesting its anti-amyloidogenic capacity. Our in vivo investigations demonstrated also that a single intracerebroventricular injection of the C60 hydrated fullerene at a dose of 7.2 nmol/ventricle significantly improved the performance of the cognitive task in control rats. The intracerebroventricular injection of the C60 hydrated fullerene (3.6 nmol/ventricle) prevented the impairment of performance of the cognitive task induced by amyloid-beta25-35 (22.5 nmol/ventricle). The results obtained may be useful in the development of therapy of Alzheimer's disease.

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Serum concentration of autoantibodies to myosin light chains was measured after resumption of the bloodflow recovery in patients who underwent hypothermic aortocoronary bypass surgery. The patients were divided into 3 groups according to postoperative hemodynamic parameters and degree of myocardial injury. The studies showed significant differences in the concentrations of autoantibodies to myosin light chains between the groups. High correlation was shown between the duration of aorta clamping and concentration of autoantibodies to myosin light chains. Some factors characterizing initial severity of the disease can modulate blood concentration of autoantibodies to myosin light chains.

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The present paper covers two series of the experiment studies performed in attempt to analyze the support-triggered cellular mechanisms, controlling the maintenance of tonic muscle fiber characteristics. Exposure to 7 day dry immersion induced significant decline of the human soleus single fiber peak isometric tension and the Ca(2+)-sensitivity of myofibrils. 30-40% losses of the relative content of titin and nebulin were found after immersion. The application of the plantar support stimulation device prevented all these alterations. In the second experimental series the treatment of hindlimb suspended rats with the Ca(2+)-binding agent (EGTA) allowed to prevent or attenuate all the above mentioned unloading-induced soleus fiber alterations. Thus it is concluded that resting Ca2+ accumulation in the unloaded fibers may be among the mechanisms involved in the changes of fiber properties during unloading.

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Artificial support stimulation is known to attenuate or prevent many motor or skeletal muscle effects of actual or simulated microgravity. The present study was purposed to analyze the effects of artificial support on human soleus fibers after 7-day exposure to supportless environment. 8 healthy male volunteers were exposed to dry immersion in supine position for 7 days according to Shulzhenko and Vil-Villiams (1972). 4 of them worn the support device which provided them with plantar stimulation in regime described elsewhere.

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Using the system of F-actin paracrystals, we have obtained electron microscopic evidence that projectin from synchronous flight muscles of Locusta migratoria binds to actin filaments in the same fashion as skeletal titin. Control actin paracrystals formed in the presence of Mg(2+) ions have great width and length and blunted ends. The addition of either projectin or titin results in disruption of compact ordered packing of F-actin in paracrystals and leads to the formation of loose filament bundles with smaller diameters and tapered ends. It is also accompanied with the appearance of individual actin filaments in considerable amounts. The effect becomes more pronounced with the increase in concentrations of added projectin or titin. Possible physiological implications of projectin-actin interactions are discussed.

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It had been hypothesized and recently shown that the exposure to gravitational unloading brought out to sufficient accumulation of Ca2+ in the myoplasm of soleus muscle fibers. Some authors believe that this dramatic Ca2+ accumulation induces the muscle protein degradation (including cytoskeletal proteins) by means of Ca 2(+)-activated proteases. For instance, the loss of giant sarcomeric cytoskeletal protein titin which is believed to determine the elasticity properties of muscle fibers, may contribute to the fiber stiffness decrease under unloading conditions. The study was designed to test the hypothesis suggesting that intracellular Ca2+ binding by means of EDTA administration would allow to attenuate hypogravity-induced atrophic changes including changes in myofibrillar proteins of skeletal muscle fibers.