IIX Myosin Heavy Chain Isoform Research Articles

Aberrant post-translational modifications compromise human myosin motor function in old age.

Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

Effects of Physical Activity and Inactivity on Muscle Fatigue

The aim of this review was to examine the mechanisms by which physical activity and inactivity modify muscle fatigue. It is well known that acute or chronic increases in physical activity result in structural, metabolic, hormonal, neural, and molecular adaptations that increase the level of force or power that can be sustained by a muscle. These adaptations depend on the type, intensity, and volume of the exercise stimulus, but recent studies have highlighted the role of high intensity, short-duration exercise as a time-efficient method to achieve both anaerobic and aerobic/endurance type adaptations. The factors that determine the fatigue profile of a muscle during intense exercise include muscle fiber composition, neuromuscular characteristics, high energy metabolite stores, buffering capacity, ionic regulation, capillarization, and mitochondrial density. Muscle fiber-type transformation during exercise training is usually toward the intermediate type IIA at the expense of both type I and IIx myosin heavy-chain isoforms. High-intensity training results in increases of both glycolytic and oxidative enzymes, muscle capillarization, improved phosphocreatine resynthesis and regulation of K+, H+, and lactate ions. Decreases of the habitual activity level due to injury or sedentary lifestyle result in partial or even compete reversal of the adaptations due to previous training, manifested by reductions in fiber cross-sectional area, decreased oxidative capacity, and capillarization. Complete immobilization due to injury results in markedly decreased force output and fatigue resistance. Muscle unloading reduces electromyographic activity and causes muscle atrophy and significant decreases in capillarization and oxidative enzymes activity. The last part of the review discusses the beneficial effects of intermittent high-intensity exercise training in patients with different health conditions to demonstrate the powerful effect of exercise on health and well being.

Correlation of dystrophin–glycoprotein complex and focal adhesion complex with myosin heavy chain isoforms in rat skeletal muscle

The dystrophin-glycoprotein complex (DGC) and focal adhesion complex (FAC) are transmembrane structures in muscle fibres that link the intracellular cytoskeleton to the extracellular matrix. DGC and FAC proteins are abundant in slow-type muscles, indicating the structural reinforcement which play a pivotal role in continuous force output to maintain posture for long periods. The aim of the present study was to examine the expression of these structures across fast-type muscles containing different myosin heavy chain (MHC) isoform patterns which reflect the fatigue-resistant characteristics of skeletal muscle. We measured the expression of dystrophin and beta1 integrin (representative proteins of DGC and FAC respectively) in plantaris, extensor digitorum longus, tibialis anterior, red and white portions of gastrocnemius, superficial portion of vastus lateralis and diaphragm, in comparison with soleus (SOL) and cardiac muscle from rats. The expression of dystrophin and beta1 integrin correlated positively with the percentage of type I, IIa and IIx MHC isoforms and negatively with that of type IIb MHC isoform in fast-type skeletal muscles, and their expression was abundant in SOL and cardiac muscle. Our results support the idea that DGC and FAC are among the factors that explain the fatigue-resistant property not only of slow-type but also of fast-type skeletal muscles.

Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression.

Rat soleus muscle consists predominantly of slow type I fibers. We have shown previously through deletion analysis that the highest level of reporter activity that we measure when injecting type I myosin heavy chain (MHC) promoter (MHC(1))-linked luciferase plasmid into soleus muscles depends on the presence of a 550-bp upstream enhancer (3,450-2,900) region of the promoter. Because the calcineurin-nuclear factor of activated T cells (NFAT) pathway has been implicated in the regulation of the slow muscle gene program, particularly the MHC(1) isoform, and the MHC(1) promoter contains several putative NFAT sites, we examined via deletion and mutation analyses whether this pathway is involved in the regulation of promoter activity in soleus. Nine days of treatment with the calcineurin inhibitor cyclosporin A (CsA) caused a significant decrease in activity of the -3,500- and -3,450-bp promoters compared with vehicle-treated rats. Truncation of the promoter to -2,900 bp or smaller reduced the activity and also eliminated the CsA responsiveness, thus implying that the enhancer region is required for CsA responsiveness. Surprisingly, mutating the two NFAT elements within the enhancer region had no obvious effect on promoter activity. CsA treatment resulted in an increase in the mRNA levels of fast-type IIa and IIx MHC isoforms, but RT-PCR analysis of MHC(1) pre-mRNA and mature mRNA expression in soleus muscles revealed no differences between vehicle- and CsA-treated rats. Although CsA affects the activity of the MHC(1) promoter, it appears that its effect is not through direct binding of NFAT to sites on the promoter.

Metabolism changes in single human fibres during brief maximal exercise.

Changes in high-energy phosphate levels in single human skeletal muscle fibres after 10 s of maximal (all-out) dynamic exercise were investigated. Muscle biopsies from vastus lateralis of two volunteers were collected at rest and immediately post exercise. Single muscle fibres were dissected from dry muscle and were assigned into one of four groups according to their myosin heavy chain (MyHC) isoform content: that is type I, IIA, IIAx and IIXa (the latter two groups containing either less or more than 50% IIX MyHC). Fragments of characterised fibres were analysed by HPLC for ATP, inosine-monophosphate (IMP), phosphocreatine (PCr) and creatine levels. After 10 s of exercise, PCr content ([PCr]) declined by approximately 46, 53, 62 and 59 % in type I, IIA, IIAx and IIXa fibres, respectively (P < 0.01 from rest). [ATP] declined only in type II fibres, especially in IIAx and IIXa fibres in which [IMP] reached mean values of 16 +/- 1 and 18 +/- 4 mmol (kg dry mass)(-1), respectively. While [PCr] was reduced in all fibre types during the brief maximal dynamic exercise, it was apparent that type II fibres expressing the IIX myosin heavy chain isoform were under a greatest metabolic stress as indicated by the reductions in [ATP].

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression.

We examined the novel interaction of hyperthyroidism and hindlimb suspension on the pattern of myosin heavy chain (MHC) expression (mRNA and protein) in skeletal muscles. Female Sprague-Dawley rats were assigned to four groups: 1) normal control (Con); 2) thyroid hormone treated [150 micrograms 3,5,3'-triiodothyronine (T3). kg-1. day-1] (T3); 3) hindlimb suspension (HS); or 4) T3-treated and HS (T3 + HS). Results show for the first time the novel observation that the combination T3 + HS induces a rapid and sustained, marked (80-90%) downregulation of type I MHC gene expression that is mirrored temporally by concomitant marked upregulation of type IIb MHC gene expression, as evidenced by the de novo synthesis of type IIb MHC protein in the soleus. The fast type IIx MHC isoform showed a differential response among the experimental groups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing in the plantaris muscles. The fast type IIa MHC was the least responsive to suspension of the MHCs and reflected its greatest responsiveness to T3 treatment while also undergoing differential adaptations in slow vs. fast muscle (increases vs. decreases, respectively). These results confirm previous findings that all four adult MHC genes are sensitive to T3 and suspension in a muscle-specific manner. In addition, we show that T3 + HS can interact synergistically to create novel adaptations in MHC expression that could not be observed when each factor was imposed separately.

ARE SKELETAL MUSCLE FIBERS OBLIGATED TO FOLLOW THE IlrarrIIAlrarrIIXlrarrIIB SCHEME OF MyHC ISOFORM PLASTICITY?

219 It has been proposed that transitions in myosin heavy chain (MyHC) isoform expression must follow an obligatory scheme summarized as follows: IlrarrIIAlrarrIIXlrarrIIB. This scheme stipulates, for instance, that slow muscle fibers can express the fast Type IIB MyHC but only after the sequential expression of the fast Type IIA and IIX MyHC isoforms. This concept was tested by using a combined treatment of hyperthyroidism and hindlimb suspension(T3+HS; n=24 animals), and comparing the MyHC isoform composition of single fibers taken from the soleus muscles in these animals with that of single fibers obtained from control animals (CON; n=24). Muscles (n=8 per group and time point) were harvested at 1, 2, and 4 wks of treatment. Approximately 40 fibers/muscle (total = 1,870 fibers) were individually isolated by microdissection, and the MyHC isoform composition of each fiber determined by electrophoresis. Combinations of MyHC isoforms found to be inconsistent with the IlrarrIIAlrarrIIXlrarrIIB scheme are shown below, and reported as a percent of the entire population of fibers for that group and time point. The major findings of this study demonstrate that: 1) the combined treatment of T3+HS creates transitions in single fiber MyHC isoform composition that are inconsistent with IlrarrIIAlrarrIIXlrarrIIB scheme of MyHC isoform plasticity; and 2) as exemplified by the I/IIB and I/IIX/IIB hybrid fibers, time-course studies are essential for accurately identifying schemes of MyHC isoform transitions at the single fiber level. TableTable

MUSCLE STRENGTH AND MYOSIN HEAVY CHAIN mRNA EXPRESSION IN THE ELDERLY AFTER WEIGHT TRAINING

1099 The purpose of this study was to determine the effects of 12 weeks of low-(LIEX) and high-intensity (HIEX) weight training on the muscular strength and myosin heavy chain (MHC) mRNA isoform expression of 16 elderly men and women using RT-PCR. Subjects were randomly assigned to either a control group (CON), or training groups of either LIEX or HIEX. Training sessions occurred 3 days/week and involved 3 sets of 12-15 repetitions at 65% 1-RM for LIEX and 3 sets of 8-10 repetitions at 85% 1-RM for HIEX. For upper-body strength, results showed increases of 6.56% for CON, and significant increases of 46.09% and 52.83%, respectively, for LIEX and HIEX. There were also increases in lower-body strength of 3.48% for CON, and significant increases of 38.18% and 55.84% for LIEX and HIEX, respectively. There were significant increases in Type IIa MHC mRNA expression for LIEX and Type I and IIa for HIEX that were significantly correlated with the respective increases in lower-body strength while the Type IIx MHC isoform in LIEX and HIEX was significantly less than CON. This study suggests that both low- and high-intensity weight training produces similar increases in strength which seem to be related to respective increases in the expression of Type I and IIa MHC mRNA isoforms in elderly men and women.

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy.

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

A new concept in laryngeal muscle: Multiple myosin isoform types in single muscle fibers of the lateral cricoarytenoid

This report describes the first known investigation of canine laryngeal muscle in which single fibers were dissected and their myosin heavy chain (MHC) isoform content was analyzed. Both SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot techniques were used. The data from single fiber SDS-PAGE indicate that the lateral cricoarytenoid (LCA) is predominantly a fast muscle composed of the following MHC isoforms: Type I, 16.3%; Type IIA, 71.3%; Type IIX, 10.4%; and Type IIB, 2.0%. The results reveal a phenomenon that, to our knowledge, has not been previously described for laryngeal muscle: the presence of two or more MHC isoforms in a single canine LCA muscle fiber. A large number (41%) of muscle fibers coexpressed two or more MHC isoforms. The three most common patterns of coexpression were Type IIA/IIX (72%), Type IIA/I (16%), and Type IIA/IIX/I (8%). Interestingly, the fast Type IIX MHC isoform was typically present with other isoforms and rarely found by itself in individual fibers. Additional experiments are underway to determine whether other laryngeal muscles exhibit such an unusually high ratio of MHC isoform polymorphism. (Otolaryngol Head Neck Surg 1998;118:86-94.)

Gender‐related differences in the regulatory influence of thyroid hormone on the expression of myosin isoforms in young and old rats

The effects of 4 weeks of thyroid hormone (3.5.3'-triiodothyronine. T3) treatment on the expression of myosin heavy chain (MyHC) isoforms were examined in young (3-6 months; and old (20-24 months) female rats, and compared with those in age-matched male rats (Larsson et al. 1995). In control rats ageing was associated with a type IIA to I MyHC isoform switching in the slow-twitch soleus and a type IIB to IIX MyHC isoform switching in the fast-twitch extensor digitorum longus muscle (EDL). Gender- and muscle-specific differences were observed in the regulation of MyHC isoforms by T3. In the soleus, dramatic downregulation of the type I and upregulation of the type IIA MyHC isoform were observed in both females and males, but upregulation of the IIX MyHC isoform was observed only in male rats. In EDL, T3 treatment had no significant influence on the MyHC isoform composition in the males irrespective of the age of the animal. In the females, on the other hand, T3 treatment resulted in a significant MyHC transformation from IIA to IIB, probably via IIX myosin, in spite of the fact that type IIA mRNA has been reported to be downregulated in both females and males. It is concluded that the regulation of MyHC isoforms by thyroid hormone differs between females and males, presumably as a result of a gender-related difference in the translational or post-translational regulation of MyHC synthesis.

Endurance training affects myosin heavy chain phenotype in regenerating fast-twitch muscle.

The aim of this study was to analyze the effects of treadmill training (2 h/day, 5 days/wk, 30 m/min, 7% grade for 5 wk) on the expression of myosin heavy chain (MHC) isoforms during and after regeneration of a fast-twitch white muscle [extensor digitorum longus (EDL)]. Male Wistar rats were randomly assigned to a sedentary (n = 10) or an endurance-trained (ET; n = 10) group. EDL muscle degeneration and regeneration were induced by two subcutaneous injections of a snake toxin. Five days after induction of muscle injury, animals were trained over a 5-wk period. It was verified that approximately 40 days after venom treatment, central nuclei were present in the treated EDL muscles from sedentary and ET rats. The changes in the expression of MHCs in EDL muscles were detected by using a combination of biochemical and immunocytochemical approaches. Compared with contralateral nondegenerated muscles, relative concentrations of types I, IIa, and IIx MHC isoforms in ET rats were greater in regenerated EDL muscles (146%, P < 0.05; 76%, P < 0.01; 87%, P < 0.01, respectively). Their elevation corresponded to a decrease in the relative concentration of type IIb MHC (-36%, P < 0.01). Although type I accounted for only 3.2% of total myosin in regenerated muscles from the ET group, the cytochemical analysis showed that the proportion of positive staining with the slow MHC antibody was markedly greater in regenerated muscles than in contralateral ones. Collectively, these results demonstrate that the regenerated EDL muscle is sensitive to endurance training and suggest that the training-induced shift in MHC isoforms observed in these muscles resulted from an additive effect of regeneration and repeated exercise.

Effect of spaceflight on skeletal muscle: mechanical properties and myosin isoform content of a slow muscle

This study examined changes in contractile, biochemical, and histochemical properties of slow antigravity skeletal muscle after a 6-day spaceflight mission. Twelve male Sprague-Dawley rats were randomly divided into two groups: flight and ground-based control. Approximately 3 h after the landing, in situ contractile measurements were made on the soleus muscles of the flight animals. The control animals were studied 24 h later. The contractile measurements included force-velocity relationship, force-frequency relationship, and fatigability. Biochemical measurements focused on the myosin heavy chain (MHC) and myosin light chain profiles. Adenosine-triphosphatase histochemistry was performed to identify cross-sectional area of slow and fast muscle fibers and to determine the percent fiber type distribution. The force-velocity relationships of the flight muscles were altered such that maximal isometric tension (Po) was decreased by 24% and maximal shortening velocity was increased by 14% (P < 0.05). The force-frequency relationship of the flight muscles was shifted to the right of the control muscles. At the end of the 2-min fatigue test, the flight muscles generated only 34% of Po, whereas the control muscles generated 64% of Po. The flight muscles exhibited de novo expression of the type IIx MHC isoform as well as a slight decrease in the slow type I and fast type IIa MHC isoforms. Histochemical analyses of flight muscles demonstrated a small increase in the percentage of fast type II fibers and a greater atrophy of the slow type I fibers. The results demonstrate that contractile properties of slow antigravity skeletal muscle are sensitive to the microgravity environment and that changes begin to occur within the 1st wk. These changes were at least, in part, associated with changes in the amount and type of contractile protein expressed.