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Objective: to assess the structural and functional state of skeletal muscles in the hip, as well as changes in the content of the sarcomere cytoskeleton proteins titin and nebulin and their phosphorylation level in patients with chronic alcohol intoxication.Patients and methods. Thirteen patients (4 men and 9 women; mean age, 38.9±9.9 years) with chronic alcohol intoxication were examined. The average duration of regular consumption of alcoholic beverages was 7.6±3.7 years. The mean amount of alcohol consumed per week was 48.2±13.1 units. A control group included 10 healthy volunteers matched for age and sex. A neurological examination was performed according to the generally accepted scheme. Laboratory tests involved blood biochemical analysis estimating the levels of liver enzymes, creatine phosphokinase (CPK), and insulin-like growth factor-1 (IGF-1). All the patients underwent hip magnetic resonance imaging (MRI), followed by assessment of the degree of muscle tissue damage and by determination of the volume of anterior and posterior thigh muscles. The content of titin and nebulin and their phosphorylation level were determined in the muscle tissue samples obtained by an open biopsy from the lateral head of the quadriceps femoris muscle.Results and discussion. Four (30.8%) patients were found to have proximal leg muscle weakness; the degree of paresis was the same in the anterior and posterior thigh muscles. There was a significant increase in the plasma level of liver enzymes; the CPK level remained within the reference values; there was a tendency towards lower IGF-1 levels. Analysis of MRI data showed that 7 (53.8%) patients had fatty degeneration in the thigh muscles. Quantitative evaluation ascertained a significant symmetrical decrease in the volume of anterior thigh muscles and a tendency towards a symmetrical reduction in that in the posterior thigh muscle compared to the control. Analysis of the content of titin and nebulin in the lateral head of the quadriceps femoris muscle revealed a significant decrease in the percentage of nebulin (81.1%; p < 0.01) and intact titin-1 (T1) isoforms (83.6%; p < 0.01). The percentage of proteolytic titin-2 (T2) fragments in the muscle of patients did not differ significantly from that in the control group. Estimating the phosphorylation level of the structural muscle proteins showed no significant differences when compared to the control.Conclusion. Anterior and posterior thigh muscle weakness should be considered as the main clinical manifestation of chronic alcoholic myopathy (CAM) in the absence of biochemical and neurophysiological markers of the disease. Lower extremity muscle MRI that can reveal a lower muscle volume concurrent with fatty degeneration is a non-invasive informative diagnostic technique for CAM. The pathogenesis of skeletal muscle atrophy in chronic alcohol intoxication involves the sarcomere structural proteins titin and nebulin, which regulate the interaction of the major contractile proteins actin and myosin, and whole muscle contraction.

Almost 40 years has passed since the discovery of giant elastic protein titin (also known as connectin) of striated and smooth muscles using gel electrophoresis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a major technique for studying the isoform composition and content of titin. This review provides historical insights into the technical aspects of the electrophoresis methods used to identify titin and its isoforms. We particularly focus on the nuances of the technique that improve the preservation of its primary structure so that its high molecular weight isoforms can be visualized. [ABSTRACT FROM AUTHOR]

Heart failure with preserved ejection fraction (HFpEF) has similar prevalence and prognosis as HF with reduced EF, but there is no approved treatment for HFpEF. HFpEF is common in postmenopausal women, which suggests that the absence of estrogen (E2) plays a role in its pathophysiology. With the country's growing elderly population, the prevalence of HFpEF is rapidly increasing. This has triggered a renewed urgency in finding novel approaches to preventing and slowing the progression of HFpEF. In this review, we address the role of E2 in left ventricular diastolic function and how it impacts women with HFpEF as well as animal models. We also discuss the primary potential mechanisms that represent critical nodes in the mechanistic pathways of HFpEF and how new treatments could be developed to target those mechanisms. [ABSTRACT FROM AUTHOR]

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction. [ABSTRACT FROM AUTHOR]

Using the real-time reverse-transcription polymerase chain reaction (RT-PCR) and low percentage SDS-gel electrophoresis, changes in the gene expression and isoform composition of a giant sarcomeric protein titin (connectin) in cardiac muscle and changes in its isoform composition in skeletal muscle m. soleus of chronically alcoholized rats were studied. The decreased content of titin protein in examined muscles and decreases in titin gene expression in the myocardium of chronically alcoholized rats were found, which indicates the development of the pathological process. [ABSTRACT FROM AUTHOR]

Circadian rhythms are approximate 24-h oscillations in physiology and behavior. Circadian rhythm disruption has been associated with increased incidence of hypertension, coronary artery disease, dyslipidemia, and other cardiovascular pathologies in both humans and animal models. Mice lacking the core circadian clock gene, brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like protein (Bmal1), are behaviorally arrhythmic, die prematurely, and display a wide range of organ pathologies. However, data are lacking on the role of Bmal1 on the structural and functional integrity of cardiac muscle. In the present study, we demonstrate that Bmal1-/- mice develop dilated cardiomyopathy with age, characterized by thinning of the myocardial walls, dilation of the left ventricle, and decreased cardiac performance. Shortly after birth the Bmal1-/- mice exhibit a transient increase in myocardial weight, followed by regression and later onset of dilation and failure. Ex vivo working heart preparations revealed systolic ventricular dysfunction at the onset of dilation and failure, preceded by downregulation of both myosin heavy chain isoform mRNAs. We observed structural disorganization at the level of the sarcomere with a shift in titin isoform composition toward the stiffer N2B isoform. However, passive tension generation in single cardiomyocytes was not increased. Collectively, these findings suggest that the loss of the circadian clock gene, Bmal1, gives rise to the development of an age-associated dilated cardiomyopathy, which is associated with shifts in titin isoform composition, altered myosin heavy chain gene expression, and disruption of sarcomere structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The present-day data on the structural and functional properties of titin in skeletal and cardiac muscle are reviewed. [ABSTRACT FROM AUTHOR]

By our modified SDS gel electrophoresis and immunoblotting, the isoform composition of titin in skeletal and cardiac muscles of human and animals was studied to reveal new titin forms above 3700 kDa in size. The data obtained suggest that the new large-size titin species are the intact (original) isoforms of this protein, whereas the known N2A, N2B, and N2BA titin bands in electrophoregrams correspond to their fragments. [ABSTRACT FROM AUTHOR]

Titin, a striated-muscle specific protein spanning the distance between Z- and M-lines of sarcomeres, is held responsible for developing passive tension and for maintaining the central position of thick filaments in contracting sarcomeres. Different muscles express titin isoforms of different molecular mass. To improve the insight in the relation between titin isoforms and kinematics of fast swimming at different ages the presence of carp larval muscle titin (Cyprinus carpio L.) was investigated and compared with data of adult carp. Gel-electrophoresis revealed that titin isoforms were larger in adult than in larval muscle. Apparently the molecular structure of titin changed during ontogeny. A previous study showed that the size of titin is correlated with the functioning of different muscles during swimming. Fish larvae (6.5-8 mm total length), subjected to low Reynolds-number regimes during swimming (Re < 500), require special features to overcome frictional effects. Fibres with smaller titin isoforms require more passive tension when being stretched. During fast swimming of larvae, passively stretched fibres at the convex side of the body axis absorb energy, generated by activity of fibres at the concave side, that is released in the successive opposite bending. [ABSTRACT FROM AUTHOR]

This paper discusses some examples of strong correlations between functions and structures in axial fish muscle on a micro-level. Muscle tissue needs a certain elasticity to cope with the diverse functional requirements necessary for swimming. During fast-starts of carp, muscles can be stretched up to 40% above their resting length. These muscles need to be designed such that they can endure these strains and simultaneously provide the required force. Posterior fibres of adult carp have a longer phase of eccentric activity (active while being stretched) than anterior ones and will therefore develop greater forces. Posterior and red fibres are subjected to larger strains during continuous swimming and have special adaptations on a microscopical level to meet these functional demands. A linear correlation was found between surface areas of myotendinous junctions (MTJs), connections between muscle fibres and collagen fibres (measure for strength) and the size and duration of load on a junction. Fibres with larger surface areas consequently can bear larger loads. Exactly those fibres subjected to larger loads during swimming, posterior and red fibres, possessed stronger MTJs. Titin and intermediate filaments are important elastic structures in muscle. Titin acts as a dual spring developing passive tension upon sarcomere stretching and shortening. Less passive tension was needed to stretch red posterior fibres, with larger titin isoforms, compared to the less elastic red anterior fibres. Intermediate filaments act as safety devices protecting muscles from permanent damage due to too high strains. Anterior fibres possessed smaller intermediate filaments, thus protecting them at shorter sarcomere lengths from being torn apart due to too large strains. This study corroborates the idea that (small) differences in muscle function during swimming, turning and escaping can be used to predict and possibly explain structural differences between muscle types and at different locations. [ABSTRACT FROM AUTHOR]

On the basis of our earlier experiments on the study of changes in the content of titin and the level of its phosphorylation in skeletal muscles that atrophied during space flight, hibernation, and because of the development of alcohol-induced lesions, it was hypothesized that an increase in the degree of titin phosphorylation results in increased proteolytic degradation of this protein, which contributes to the development of skeletal muscle atrophy. [ABSTRACT FROM AUTHOR]

Titin, a giant sarcomeric protein, is involved in the generation of passive tension during muscle contraction, assembly and stability of the sarcomere in striated muscles. Titin gene produces numerous titin protein isoforms with different sizes (∼3-4 MDa) resulting from alternative splicing. To study titin and titin isoform changes under disease conditions, the method to detect and quantify titin protein isoforms is needed. The method reported here is a 1% vertical SDS-agarose gel electrophoresis system that can solubilize, detect and quantify various titin isoform sizes. Sodium dodecyl sulfate (SDS)-agarose gel electrophoresis is an important tool in revealing the size and quantity of giant proteins in the sarcomere. In this method article, heart tissues were dissolved in urea-thiourea-glycerol sample buffer. Muscle proteins were resolved on 1% SDS-agarose gels that were silver-stained subsequently. Titin isoform bands with different sizes were separated on the gel. At the end, we also validated the method for large protein detection. Our results indicated that this electrophoresis method is efficient to study the transitions in titin isoforms. •This method provides efficient protein extraction with urea-thiourea-glycerol buffer from hard tissues such as striated muscles•This method provides an efficient way to separate large proteins over 500 kDa•Combining with silver staining, our method can detect large protein isoforms and quantify the separated protein bands.