Changes in myosin heavy chain isoforms during chronic low-frequency stimulation of rat fast hindlimb muscles. A single-fiber study

Fast-twitch rat muscles contain three fast myosin heavy chains (HC) which can be separated by density gradient gel electrophoresis. Their mobility increases in the order of HCIIa less than HCIId less than HCIIb. In contrast to the rabbit, where chronic low-frequency nerve stimulation induces a fast-to-slow conversion, stimulation for up to 56 days does not lead to appreciable increases in the relative concentration of the slow myosin heavy chain HCI in rat fast-twitch muscles. However, chronic stimulation of rat fast-twitch muscle does evoke a rearrangement of the fast myosin heavy chain isoform pattern with a progressive decrease in HCIIb and progressive increases in HCIIa and HCIId. As judged from the time course and extent of these transitions, it appears that HCIId is an intermediate form between HCIIb and HCIIa. Single-fiber analyses of normal muscles make it possible to assign these heavy chain isoforms to histochemically defined fiber types IIB, IID, and IIA. The stimulation-induced fiber transformations produce numerous hybrid fibers displaying more than one myosin heavy chain isoform. Some transforming fibers contain up to four different myosin heavy chain isoforms.     

Changes in myosin heavy-chain isoform synthesis of chronically stimulated rat fast-twitch muscle.

Chronic low-frequency stimulation was used for studying the adaptive potential of rat fast-twitch muscle to increased neuromuscular activity. The sequential exchange of myosin heavy chain isoforms HCIIb with HCIId and HCIIa was studied at the translational level using an in-vivo-labeling technique with [35S]methionine. Alterations in heavy chain isoform synthesis, i.e. a decrease in the labeling of HCIIb concomitant with an enhanced labeling of HCIId/IIa, were detectable already two days after the onset of stimulation. This time course corresponds to the previously observed alterations in the amounts of HCIIb and HCIIa mRNAs. However, significant changes in the relative protein amounts of HCIIb and HCIId/IIa were recorded only after an 8-day stimulation period. This delay at the protein level was interpreted to relate to the slow turnover of HCIIb which was estimated from its decay in long-term stimulated muscles with an approximate value of 14.7 days. Therefore, protein degradation seems to be an important post-translational regulatory step in the remodeling process of the thick filament.     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Summary Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).     

Changes in myosin heavy chain isoform expression of overloaded rat skeletal muscles

• 1.1. The effect of functional overload produced by tenotomy of synergistic gastrocnemius muscle on the expression of myosin heavy chain (MHC) isoforms in the plantaris and soleus muscles of the rat was studied using gradient sodium dodecyl sulfate-acrylamide gel electrophoresis.
• 2.2. Five weeks tenotomy, the plantaris and soleus muscle weights induced by tenotomy of the gastrocnemius muscle were 44.3% (P < 0.005) and 37.4% (P < 0.005), respectively, heavier than the contralateral control muscles.
• 3.3. Although four types of MHC isoforms were observed in both control and experimental plantaris, the percentage of MHC isoforms in the control and experimental muscles differed; the hypertrophied plantaris muscle contained more HCI (P < 0.05), HCIIa and HCIId (P < 0.05) and less HCIIb (P < 0.05) than the control muscle.
• 4.4. The control soleus muscle contained two MHC isofonns, HCI and HCIIa. However, there was only a single HCI isoform in the hypertrophied soleus muscle.
• 5.5. These results indicate that overloading a skeletal muscle by removing its synergists produces not only the muscle hypertrophy but also the changes in the expression of MHC isofonns.

     

Regional differences in myosin heavy chain isoforms and enzyme activities of the rat diaphragm.

Myosin heavy chain isoforms and enzyme activities were compared between the costal and crural regions of the rat diaphragm. The percentage of heavy chain (HC) IIb in the crural region of the diaphragm was significantly (P less than 0.05) higher than that in the costal region (mean 7.3 vs. 3.0%), and the percentage of HCI was significantly lower in the crural than in the costal diaphragm (22.7 vs. 27.9%). The distributions of HCIIa and HCIId were relatively homogeneous in both regions. Succinate dehydrogenase activity in the costal diaphragm was 21% greater (P less than 0.01) than in the crural diaphragm. In contrast, there was no significant difference in the activity of phosphofructokinase in the crural and costal diaphragms. These results demonstrate that a difference in myosin heavy chain isoforms and oxidative capacity exists between the costal and crural regions of the rat diaphragm.     

The multiplicity of combinations of myosin light chains and heavy chains in histochemically typed single fibres. Rabbit tibialis anterior muscle.

1. Combined histochemical and biochemical single-fibre analyses [Staron & Pette (1987) Biochem. J. 243, 687-693], were used to investigate the rabbit tibialis-anterior fibre population. 2. This muscle is composed of four histochemically defined fibre types (I, IIC, IIA and IIB). 3. Type I fibres contain slow myosin light chains LC1s and LC2 and the slow myosin heavy chain HCI, and types IIA and IIB contain the fast myosin light chains LC1f, LC2f and LC3f and the fast heavy chains HCIIa and HCIIb respectively. 4. A small fraction of fibres (IIAB), histochemically intermediate between types IIA and IIB, contain the fast light myosin chains but display a coexistence of HCIIa and HCIIb. 5. Similarly to the soleus muscle, C fibres in the tibialis anterior muscle contain both fast and slow myosin light chains and heavy chains. The IIC fibres show a predominance of the fast forms and the IC fibres (histochemically intermediate between types I and IIC) a predominance of the slow forms. 6. A total of 60 theoretical isomyosins can be derived from these findings on the distribution of fast and slow myosin light and heavy chains in the fibres of rabbit tibialis anterior muscle.     

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.     

Correlation between myofibrillar ATPase activity and myosin heavy chain composition in rabbit muscle fibers

Summary Combined histochemical and biochemical analyses were performed on single fibers of rabbit soleus muscle. Histochemically, four fiber types (I, IC, IIC, IIA) were defined. Of these, types I and IIA were separate, histochemically homogeneous groups. A heterogeneous C fiber population exhibited a continuum of staining intensities between types I and IIA. Microelectrophoretic analyses of specific, histochemically defined fibers revealed that type I fibers contained exclusively HCI, whereas type IIA fibers contained only HCIIa. The C fibers were characterized by the coexistence of both heavy chains in varying ratios, type HC with a predominance of HCI and type IIC with a predominance of HCIIa. A direct correlation existed between the myosin heavy chain composition and the histochemical mATPase staining and was especially evident in the C fiber population with its variable HCI/HCIIa ratio. This correlation did not apply to the myosin light chain complement.     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Changes in milk performance and hepatic metabolism in mid-lactating dairy goats after being fed a high concentrate diet for 10 weeks

Feeding a high concentrate (HC) diet is a widely used strategy for supporting high milk yields, yet it may cause certain metabolic disorders. This study aimed to investigate the changes in milk production and hepatic metabolism in goats fed different proportions of concentrate in the diet for 10 weeks. In total, 12 mid-lactating goats were randomly assigned to an HC diet (65% concentrate of dry matter, n=6) or a low concentrate (LC) diet (35% concentrate of dry matter, n=6). Compared with LC, HC goats produced greater amounts of volatile fatty acids and produced more milk and milk lactose, fat and protein (P<0.01). HC goats showed a greater concentration of ATP, NAD, plasma non-esterified fatty acids and hepatic triglycerides than LC goats (P<0.05). Real-time PCR results showed that messenger RNA (mRNA) expression of gluconeogenic genes, namely, glucose-6-phosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase were significantly up-regulated and accompanied greater gluconeogenic enzyme activities in the liver of HC goats. Moreover, the expression of hepatic lipogenic genes including sterol regulatory element-binding protein 1c, fatty acid synthase and diacylglycerol acyltransferase mRNA was also up-regulated by the HC diet (P<0.05). HC goats had greater hepatic phosphorylation of AMP-activated protein kinase than LC (P<0.05). Furthermore, histone-3-lysine-27-acetylation contributed to this elevation of gluconeogenic gene expression. These results indicate that lactating goats fed an HC diet for 10 weeks produced more milk, which was associated with up-regulated gene expression and enzyme activities involved in hepatic gluconeogenesis and lipogenesis.     

Distinct vascular and intestinal smooth muscle myosin heavy chain mRNAs are encoded by a single-copy gene in the chicken

We examined whether the gizzard MHC gene is expressed in other smooth muscle tissues and, if so, whether there exist any smooth muscle MHC isoforms at the mRNA level. Northern blot analysis showed that the gizzard MHC gene was also expressed in the aorta and jejunum, but not in the pectoralis muscle or in fibroblasts. This indicates that striated muscle and non-muscle MHC isoforms are encoded in genes distinct from the smooth muscle MHC gene. Further, nuclease S1 mapping showed that the aortic smooth muscle MHC mRNA was distinct from the gizzard mRNA in the 5'-terminal coding region. Both of these mRNA species are expressed in the jejunum. These observations suggest that there exist at least two chicken smooth muscle MHC isoforms, vascular-type and intestinal-type, and that these isoforms are generated from a single-copy gene, probably by an alternative mRNA processing mechanism.