Regulation of myosin heavy-chain gene expression during skeletal-muscle hypertrophy.

Changes in the myosin phenotype of differentiated muscle are a prominent feature of the adaptation of the tissue to a variety of physiological stimuli. In the present study the molecular basis of changes in the proportion of myosin isoenzymes in rat skeletal muscle which occur during compensatory hypertrophy caused by the combined removal of synergist muscles and spontaneous running exercise was investigated. The relative amounts of sarcomeric myosin heavy (MHC)- and light (MLC)-chain mRNAs in the plantaris (fast) and soleus (slow) muscles from rats was assessed with cDNA probes specific for different MHC and MLC genes. Changes in the proportion of specific MHC mRNA levels were in the same direction as, and of similar magnitude to, changes in the proportion of myosin isoenzymes encoded for by the mRNAs. No significant changes in the proportion of MLC proteins or mRNA were detected. However, high levels of MLC3 mRNA were measured in both normal and hypertrophied soleus muscles which contained only trace amounts of MLC3 protein. Small amounts of embryonic and neonatal MHC mRNAs were induced in both muscles during hypertrophy. We conclude that the change in the pattern of myosin isoenzymes during skeletal-muscle adaptation to work overload is a consequence of changes in specific MHC mRNA levels.     

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

We examined the novel interaction ofhyperthyroidism and hindlimb suspension on the pattern of myosin heavychain (MHC) expression (mRNA and protein) in skeletal muscles. FemaleSprague-Dawley rats were assigned to four groups:1) normal control (Con);2) thyroid hormone treated[150 µg 3,5,3'-triiodothyronine(T₃) · kg¹ · day¹](T₃);3) hindlimb suspension (HS); or4)T₃-treated and HS(T₃ + HS). Results show for thefirst time the novel observation that the combinationT₃ + HS induces a rapid andsustained, marked (80-90%) downregulation of type I MHC geneexpression that is mirrored temporally by concomitant markedupregulation of type IIb MHC gene expression, as evidenced by the denovo synthesis of type IIb MHC protein in the soleus. The fast type IIxMHC isoform showed a differential response among the experimentalgroups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing inthe plantaris muscles. The fast type IIa MHC was the least responsiveto suspension of the MHCs and reflected its greatest responsiveness toT₃ treatment while also undergoingdifferential adaptations in slow vs. fast muscle (increases vs.decreases, respectively). These results confirm previous findings thatall four adult MHC genes are sensitive toT₃ and suspension in amuscle-specific manner. In addition, we show thatT₃ + HS can interactsynergistically to create novel adaptations in MHC expression thatcould not be observed when each factor was imposed separately.

     

Different effects on human skeletal myosin heavy chain isoform expression: strength vs. combination training.

Myosin heavy chain (MHC) isoform expression changes with physical training. This may be one of the mechanisms for muscular adaptation to exercise. We aimed to investigate the effects of different strength-training protocols on MHC isoform expression, bearing in mind that alpha- MHC(slow) (newly identified MHC isoform) mRNA may be upregulated in response to training. Twelve volunteers performed a 6-wk strength training with maximum contractions (Max group), and another 12 of similar age performed combination training of maximum contractions and ballistic and stretch-shortening movements (Combi group). Muscle samples were taken from triceps brachii before and after training. MHC isoform composition was determined by SDS-PAGE silver staining, and mRNA levels of MHC isoforms were determined by RT-PCR. In Max group, there was an increase in MHC(2A) (49.4 to 66.7%, P < 0.01) and a decrease in MHC(2X) (33.4 to 19.5%, P < 0.01) after training, although there was no significant change in MHC(slow). In Combi group, there was also an increase in MHC(2A) (47.7 to 62.7%, P < 0.05) and a decrease in MHC(slow) (18.2 to 9.2%, P < 0.05) but no significant change in MHC(2X). An upregulation of alpha-MHC(slow) mRNA was, therefore, found in both groups as a result of training. The strength training with maximum contractions led to a shift in MHC isoform composition from 2X to 2A, whereas the combined strength training produced an MHC isoform composition shift from slow to 2A.     

Changes in myosin isozyme expression during cardiac hypertrophy in hyperthyroid rabbits

The myosin isozyme distribution in the left ventricle and in the interventricular septum of rabbits was studied after 3, 7, 11, 14 and 21 days of L-thyroxine (500 micrograms/kg/day) administration. Histochemical procedures were employed to identify V1 and V3 by their Ca2+ ATPase activity and their proportions were quantified through polyacrylamide gel electrophoresis. In the left ventricle, the subepicardium was the first to show the shift from V3 to V1, followed by the subendocardium. The intermediate region became heterogeneous by 11 days and remained so until 21 days. The right subendocardial and the intermediate regions of the interventricular septum were heterogeneous in the normal rabbit and hyperthyroidism resulted in a shift from V3 to V1 in both the right and left subendocardial regions of the septum. Like the left ventricle, the intermediate region of the interventricular septum remained heterogeneous. Localized accumulations of collagen were seen in all regions of the left ventricle and interventricular septum. From these results we conclude that in thyrotoxic myocardial hypertrophy the isozymic shift from V3 to V1 is progressive, region-specific and is directly correlated with the period of hyperthyroidism in the first 2 weeks. Prolonged hyperthyroidism results in localized accumulation of collagen which does not exhibit any regional specificity.     

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

The relationshipbetween myogenin or MyoD expression and hypertrophy of the rat soleusproduced either by clenbuterol and 3,3',5-triiodo-L-thyronine(CT) treatment or by surgical overload was examined. Mature female ratswere subjected to surgical overload of the right soleus with the leftsoleus serving as a control. Another group received the same surgicaltreatment but were administered CT. Soleus muscles were harvested 4 wkafter surgical overload and weighed. Myosin heavy chain isoforms wereseparated by using polyacrylamide gel electrophoresis while myogeninand MyoD expression were evaluated by Northern analysis.CT and functional overload increased soleus muscle weight. CT treatmentinduced 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 inCT-treated or non-CT-treated rats. Thus pharmacologically andsurgically induced hypertrophy have differing effects on myogenin andMyoD expression, because their levels were associated with changes inmyosin heavy chain composition (especially type IIX) rather thanchanges in muscle mass.

     

Changes in skeletal-muscle myosin isoenzymes with hypertrophy and exercise.

The patterns of myosin isoenzymes in fast- and slow-twitch muscles of the rat hindlimb were studied, by pyrophosphate/polyacrylamide-gel electrophoresis, with hypertrophy (induced by synergist removal) and with spontaneous running exercise of 4 and 11 weeks duration. At 11 weeks, changes with hypertrophy in the slow-twitch soleus, composed of greater than 95% SM2 (slow myosin 2) in normal muscles, were minor, and consisted of an increase in the SM1 and SM1', and a loss of intermediate myosin (IM), an isoenzyme characteristic of Type IIa fibres [Fitzsimons & Hoh (1983) J. Physiol. (London) 343, 539-550]. The changes were dramatic, however, in the fast-twitch plantaris muscle. There was a 3-fold increase in the proportion of SM. In addition, IM became the predominant isoenzyme in the profile of hypertrophied plantaris by 4 weeks. These increases were balanced by decreases in the proportion of FM2 (fast myosin 2), with FM1 completely absent from the profile at 11 weeks. The changes in the plantaris with exercise were similar in direction but not as extensive as those with hypertrophy, and FM1 remained present at control levels throughout the study. When hypertrophy and exercise were combined, the increase in slow myosin was equal to the sum of the increases with each treatment alone. Changes at 4 weeks were intermediate between those of control and 11-week muscles. Peptide mapping of individual myosin isoenzymes showed that the heavy chains of IM were different from either fast or slow heavy chains. Furthermore, IM was found to be composed of a mixture of fast and slow light chains. These changes suggest that a transformation of myosin from fast to slow isoforms was in progress in the plantaris in response to hypertrophy, via a Type-IIa-myosin (IM) intermediate stage, a phenomenon similar to that occurring in chronically stimulated fast muscles during fast-to-slow transformation [Brown, Salmons & Whalen (1983) J. Biol. Chem. 258, 14686-14692].     

Competitive inhibition of myosin ATPase activity by different molecular weight heparins.

Myosin ATPase activity was measured, by continuous luminometric method, in presence of different molecular weight heparins. ATPase activity decreases in the presence of heparin, when simultaneous incubation with ATP is carried out; the percentage of inhibition is proportional to polysaccharide concentration. Heparins of different molecular weights (1.75 KD to 11.6 KD) are competitive inhibitors of enzymatic activity; the inhibitory effects is also appreciable with trisulphated disaccharide. The possible mechanisms of interaction between heparin and myosin ATPase are discussed.     

Age effects on myosin subunit and biochemical alterations with skeletal muscle hypertrophy.

The purpose of this study was to determine whether skeletal muscle mass, myofibrillar adenosinetriphosphatase activity, and the expression of myosin heavy (MHC) and light chain subunits are differentially affected in juvenile (4 wk) and young adult (12 wk) rats by a hypertrophic growth stimulus. Hypertrophy of the plantaris or soleus was studied 4 wk after ablation of either two [gastrocnemius (GTN) and soleus or plantaris] or one (GTN) synergistic muscle(s). There was no difference in the relative magnitude of hypertrophy because of age. Plantaris myofibrillar adenosinetriphosphatase activity was decreased 21 and 12% in juvenile and adult rats, respectively, as a result of ablation of both the GTN and soleus. Slow myosin light chain isoforms (1s and 2s) were expressed to a greater extent in hypertrophied plantaris muscles of both ages, but the increase in 1s was greater in juvenile rats. The relative expression of slow beta-MHC in hypertrophied plantaris muscles increased by 470 and 350%, whereas MHC IIb decreased by 70 and 33% in juvenile and adult rats, respectively. The relative expression of MHC IIa increased (56%) in the plantaris after ablation in juvenile rats only. These shifts in myosin subunit expression and the increases in mass were generally about one-half the magnitude when only the GTN was removed. There were no detectable myosin shifts in hypertrophied soleus muscles. Although the extent of muscle hypertrophy is similar, the shifts in myosin subunits were greater in juvenile than in young adult rats.     

Proper expression of myosin genes in transgenic nematodes.

Caenorhabditis elegans has four genes which encode skeletal myosin heavy chain isoforms. We have re-introduced clones of two of these genes, myo-3 and unc-54 at low copy number into the germline of C. elegans. The resulting loci behave as functional copies of the genes by two genetic criteria: (i) they can result in phenotypic rescue of strains carrying inactivating myo-3 or unc-54 mutations, and (ii) their presence in strains with wild-type copies of the endogenous myosin loci has genetic consequences similar to duplicating the endogenous loci. The re-introduced genes function at a level close to that of the endogenous loci. Monoclonal antibodies specific for the different isoforms have been used to localize the expressed proteins. The re-introduced genes express in precisely the same cell types as the endogenous genes, and the myosin products produced assemble into filament structures as in wild-type. Unexpectedly, we have found in the course of this work that very high copy numbers of the unc-54 gene lead to a disruption of muscle structure which may result from overexpression of the protein product.     

Actin and myosin: control of filament assembly

Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.     

Competitive/co-operative interactions in acid base sandwich: role of cation vs. substituents

Journal of Molecular Modeling - The glass transition temperature (T g) and density of poly-(phthalazinone ether sulfone ketone) (PPESK A) were estimated by molecular dynamic (MD) simulation. A...