Slow Isoform Research Articles

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type-specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin-deficient double-muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse-transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC-2x and -2a and less -slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster-type muscles and on MyoD expression in slower-type muscles, suggesting a possible muscle type-specific effect of myostatin in skeletal muscle growth and maintenance.

Loss of ovarian function in mice results in abrogated skeletal muscle PPARδ and FoxO1-mediated gene expression

Menopause, the age-related loss of ovarian hormone production, promotes increased adiposity and associated metabolic pathology, but molecular mechanisms remain unclear. We previously reported that estrogen increases skeletal muscle PPARδ expression in vivo, and transgenic mice overexpressing muscle-specific PPARδ are reportedly protected from diet-induced obesity. We thus hypothesized that obesity observed in ovariectomized mice, a model of menopause, may result in part from abrogated expression of muscle PPARδ and/or downstream mediators such as FoxO1. To test this hypothesis, we ovariectomized (OVX) or sham-ovariectomized (SHM) 10-week old female C57Bl/6J mice, and subsequently harvested quadriceps muscles 12 weeks later for gene expression studies. Compared to SHM, muscle from OVX mice displayed significantly decreased expression of PPARδ (3.4-fold), FoxO1 (4.5-fold), PDK-4 (2.3-fold), and UCP-2 (1.8-fold). Consistent with studies indicating PPARδ and FoxO1 regulate muscle fiber type, we observed dramatic OVX-specific decreases in slow isoforms of the contractile proteins myosin light chain (11.1-fold) and troponin C (11.8-fold). In addition, muscles from OVX mice expressed 57% less myogenin (drives type I fiber formation), 2-fold more MyoD (drives type II fiber formation), and 1.6-fold less musclin (produced exclusively by type II fibers) than SHM, collectively suggesting a shift towards less type I oxidative fibers. Finally, and consistent with changes in PPARδ and FoxO1 activity, we observed decreased expression of atrogin-1 (2.3-fold) and MuRF-1 (1.9-fold) in OVX mice. In conclusion, muscles from ovariectomized mice display decreased PPARδ and FoxO1 expression, abrogated expression of downstream targets involved in lipid and protein metabolism, and gene expression profiles indicating less type I oxidative fibers.

Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force–frequency curve. This rightward shift of the force–frequency curve could not be explained by altered sarcoplasmic reticulum Ca2+ handling, as indicated by analysis of Ca2+ transients in intact myofibers and expression of Ca2+-handling proteins, but can be explained by the reduced myofilament Ca2+ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca2+ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.

Skeletal muscle gene expression after myostatin knockout in mature mice

There is much interest in developing anti-myostatin agents to reverse or prevent muscle atrophy in adults, so it is important to characterize the effects of reducing myostatin activity after normal muscle development. For assessment of the effect of loss of myostatin signaling on gene expression in muscle, RNA from mice with postdevelopmental myostatin knockout was analyzed with oligonucleotide microarrays. Myostatin was undetectable in muscle within 2 wk after Cre recombinase activation in 4-month-old male mice with floxed myostatin genes. Three months after myostatin depletion, muscle mass had increased 26% (vs. 2% after induction of Cre activity in mice with normal myostatin genes), at which time the expression of several hundred genes differed in knockout and control mice at nominal P < 0.01. In contrast to previously reported effects of constitutive myostatin knockout, postdevelopmental knockout did not downregulate expression of genes encoding slow isoforms of contractile proteins or genes encoding proteins involved in energy metabolism. Several collagen genes were expressed at 20-50% lower levels in the myostatin-deficient muscles, which had approximately 25% less collagen than normal muscles as reflected by hydroxyproline content. Most of the other genes affected by myostatin depletion have not been previously linked to myostatin signaling. Gene set enrichment analysis suggested that Smads are not the only transcription factors with reduced activity after myostatin depletion. These data reinforce other evidence that myostatin regulates collagen production in muscle and demonstrate that many of the previously reported effects of constitutive myostatin deficiency do not occur when myostatin is knocked out in mature muscles.

Thermal preconditioning prevents fiber type transformation of the unloading induced-atrophied muscle in rats

Muscle atrophy is accompanied by a slow-to-fast transformation of the slow muscle, e.g., the soleus muscle, which is characterized by a decrease in the expression of the slow myosin heavy chain (MyHC) isoform. Heat stress before hindlimb unloading, i.e., thermal preconditioning, has been shown to reduce the rate of disuse-induced muscle atrophy. The present study examined whether thermal preconditioning could prevent a slow-to-fast transformation of the MyHC isoform through the induction of heat-shock protein (HSP) 72. Thermally preconditioned rats (Heat + HU) were individually placed in an environmentally controlled heat chamber for 1 h before hindlimb unloading for 2 weeks (HU). Although the mean fiber cross-sectional areas of the soleus muscle decreased in the HU and Heat + HU group, the loss of myofibrillar protein was attenuated in the Heat + HU group. Furthermore, a slow-to-fast transformation of MyHC isoform was inhibited in the Heat + HU group with the overexpression of HSP72. These results indicate that thermal preconditioning before hindlimb unloading attenuates the decrease of the slow MyHC isoform in the soleus muscle. Therefore, thermal preconditioning provides a new approach to prevent disuse-induced fiber type transformation of skeletal muscle.

Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.

Comparison of cloned and non-cloned Holstein heifers in muscle contractile and metabolic characteristics

Muscle contractile and metabolic characteristics were studied on nine cloned and eight non-cloned (control) heifers. The animals were submitted to repeated biopsies of the semitendinosus (ST) muscle at the ages of 8, 12, 18 and 24 months. The contractile type was determined from the proportion of the different myosin heavy chain (MyHC) isoforms separated by electrophoresis. Glycolytic metabolism was assessed by lactate dehydrogenase (LDH) activity, and oxidative metabolism was assessed by isocitrate dehydrogenase (ICDH), cytochrome-c oxidase (COX) and β-hydroxyacyl-CoA dehydrogenase (HAD) activities. In cloned heifers at 8 months of age, there was a greater proportion of MyHC I (slow oxidative isoform) and MyHC IIa (fast oxido-glycolytic isoform), a lower proportion of MyHC IIx (fast glycolytic isoform), greater COX and HAD activity and a lower LDH/ICDH ratio compared with control heifers. Thus, young cloned heifers had slower muscle types associated with a more oxidative muscular metabolism than control heifers. From 12 months of age onwards, no significant differences were observed between cloned and control heifers. A delay in muscle differentiation and maturation in cloned heifers is hypothesised and discussed.

Fiber Type Composition of Cadaveric Human Rotator Cuff Muscles

Descriptive cadaveric laboratory study. To identify the fiber type composition of the rotator cuff and teres major muscles in human subjects. The rotator cuff is commonly injured in athletics and is a major focus of sports medicine. Although the anatomy and architecture of each muscle have been described in great detail, these muscles have never been fiber typed using immunohistochemistry or gel electrophoresis. Fiber typing is important in modeling function, exercise training, and rehabilitation. We harvested tissue samples for all 4 rotator cuff muscles, as well as the teres major muscle from cadavers. Tissues were frozen in liquid nitrogen and sectioned. Cryosections were labeled with commercially available antibodies against fast and slow isoforms of myosin heavy chain (MHC). We also harvested fresh (unembalmed) tissue from deceased subjects and labeled tissue sections with antibodies against fast or slow MHC and wheat germ agglutinin. Gel electrophoresis followed by silver staining was also used to identify and quantify MHC isoforms in fresh tissue samples. All of the muscles were of mixed fiber type composition. As a whole, 44% of rotator cuff fibers labeled positively for slow MHC, with slow MHC content of 54% in supraspinatus, 41% in infraspinatus, 49% in teres minor, 38% in subscapularis, and 40% in teres major. Mixed MHC isoform distribution was confirmed by SDS-PAGE, which also indicated that the IIa and IIx isoforms were roughly equally present across the muscles. Human rotator cuff muscles, at least in older subjects, have a mixed fiber type. Because we only examined older subjects, we must limit our interpretation to this population.

Skeletal Muscle-Specific Ablation of raptor, but Not of rictor, Causes Metabolic Changes and Results in Muscle Dystrophy

Mammalian target of rapamycin (mTOR) is a central controller of cell growth. mTOR assembles into two distinct multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. Here we show that the mTORC1 component raptor is critical for muscle function and prolonged survival. In contrast, muscles lacking the mTORC2 component rictor are indistinguishable from wild-type controls. Raptor-deficient muscles become progressively dystrophic, are impaired in their oxidative capacity, and contain increased glycogen stores, but they express structural components indicative of oxidative muscle fibers. Biochemical analysis indicates that these changes are probably due to loss of activation of direct downstream targets of mTORC1, downregulation of genes involved in mitochondrial biogenesis, including PGC1alpha, and hyperactivation of PKB/Akt. Finally, we show that activation of PKB/Akt does not require mTORC2. Together, these results demonstrate that muscle mTORC1 has an unexpected role in the regulation of the metabolic properties and that its function is essential for life.

The role of L-type calcium channels in the accumulation of Ca2+ in soleus muscle fibers in the rat and changes in the ratio of myosin and serca isoforms in conditions of gravitational unloading

Gravitational unloading is known to produce changes in the expression of a number of contractile and regulatory proteins in the soleus muscle. This applies particularly to isoforms of myosin heavy chains (MHC) and SERCA sarcoplasmic reticulum calcium pumps. Unloading increases the resting levels of extracellular calcium in soleus muscle fibers. The present study addresses verification of the hypothesis that changes in the expression of MHC and SERCA isoforms in gravitational unloading are linked with the accumulation of calcium ions in the myoplasm of muscle fibers. It is suggested that specific blockade of L-type calcium channels using nifedipine decreases the myoplasmic calcium ion concentration, thus preventing the development of changes in the expression of MHC and SERCA isoforms. A total of 36 male Wistar rats were divided into three groups: a control group, an unloading group using the Morley-Holton soleus muscle functional unloading model, and an unloading + nifedipine group, where animals received daily nifedipine (7 mg/kg/day) with their drinking water on the background of suspension. The results showed that blockade of L-type calcium channels on the background of gravitational unloading significantly decreased the extent of calcium ion accumulation in the myoplasm of soleus muscle fibers, which partly prevented the transformation of muscle fibers (in relation to the fast and slow isoforms of MHC and SERCA) to the rapid type. There was no nuclear translocation of the greater part of transcription factor NFATc1, as seen on unloading.

Influence of fast and slow alkali myosin light chain isoforms on the kinetics of stretch-induced force transients of fast-twitch type IIA fibres of rat

This study contributes to understand the physiological role of slow myosin light chain isoforms in fast-twitch type IIA fibres of skeletal muscle. These isoforms are often attached to the myosin necks of rat type IIA fibres, whereby the slow alkali myosin light chain isoform MLC1s is much more frequent and abundant than the slow regulatory myosin light chain isoform MLC2s. In the present study, single-skinned rat type IIA fibres were maximally Ca(2+) activated and subjected to stepwise stretches for causing a perturbation of myosin head pulling cycles. From the time course of the resulting force transients, myosin head kinetics was deduced. Fibres containing MLC1s exhibited slower kinetics independently of the presence or absence of MLC2s. At the maximal MLC1s concentration of about 75%, the slowing was about 40%. The slowing effect of MLC1s is possibly due to differences in the myosin heavy chain binding sites of the fast and slow alkali MLC isoforms, which changes the rigidity of the myosin neck. Compared with the impact of myosin heavy chain isoforms in various fast-twitch fibre types, the influence of MLC1s on myosin head kinetics of type IIA fibres is much smaller. In conclusion, the physiological role of fast and slow MLC isoforms in type IIA fibres is a fine-tuning of the myosin head kinetics.

Molecular basis of the phenotypic characteristics of mammalian muscle fibres.

Adult mammalian skeletal muscle fibres can be separated into two distinct groups, fast and slow. Within each group there is a continuum of metabolic enzyme activity levels. In addition there are fast and slow isoforms of various myofibrillar proteins such as myosin, tropomyosin and troponin. These proteins are multimeric and multiple isoforms of their subunits assemble to create a continuum of subtypes within each major group. Fibres which coexpress both fast and slow subunit isoforms have an increased number of possible isoform combinations such that an entire spectrum of fibre 'types' is found between the two extremes, fast and slow. Numerous myosin heavy chain and fast troponin T isoforms further increase the diversity of muscle fibres. Such cellular diversity helps to explain the dynamic nature of skeletal muscle. Each individual fibre is able to respond to various functional demands by appropriate changes in its phenotypic expression of specific proteins.

Kinetic Analysis of the Slow Skeletal Myosin MHC-1 Isoform from Bovine Masseter Muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as β-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin–ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant KD) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (KAD/KD∼5 instead of KAD/KD∼50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s−1) than reported previously (138 s−1). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.

The Slow Skeletal Muscle Isoform of Myosin Shows Kinetic Features Common to Smooth and Non-muscle Myosins

Fast and slow mammalian muscle myosins differ in the heavy chain sequences (MHC-2, MHC-1) and muscles expressing the two isoforms contract at markedly different velocities. One role of slow skeletal muscles is to maintain posture with low ATP turnover, and MHC-1 expressed in these muscles is identical to heavy chain of the beta-myosin of cardiac muscle. Few studies have addressed the biochemical kinetic properties of the slow MHC-1 isoform. We report here a detailed analysis of the MHC-1 isoform of the rabbit compared with MHC-2 and focus on the mechanism of ADP release. We show that MHC-1, like some non-muscle myosins, shows a biphasic dissociation of actin-myosin by ATP. Most of the actin-myosin dissociates at up to approximately 1000 s(-1), a very similar rate constant to MHC-2, but 10-15% of the complex must go through a slow isomerization (approximately 20 s(-1)) before ATP can dissociate it. Similar slow isomerizations were seen in the displacement of ADP from actin-myosin.ADP and provide evidence of three closely related actin-myosin.ADP complexes, a complex in rapid equilibrium with free ADP, a complex from which ADP is released at the rate required to define the maximum shortening velocity of slow muscle fibers (approximately 20 s(-1)), and a third complex that releases ADP too slowly (approximately 6 s(-1)) to be on the main ATPase pathway. The role of these actin-myosin.ADP complexes in the mechanochemistry of slow muscle contraction is discussed in relation to the load dependence of ADP release.

Regulation of troponin T expression during muscle development in sea breamSparus auratusLinnaeus: the potential role of thyroid hormones

In the sea bream Sparus auratus three stage-specific fast troponin T (fTnT) isoforms have been cloned and correspond to embryonic-, larval- and adult-specific isoforms. Characterisation, using database searches, of the putative genomic organisation of Fugu rubripes and Tetraodon nigroviridis fTnT indicates that alternative exon splicing in the 5 region of the gene generates the different isoforms. Moreover, comparison of teleost fTnTs suggests that alternative splicing of fTnT appears to be common in teleosts. A different temporal expression pattern for each fTnT splice varotnt is found during sea bream development and probably relates to differing functional demands, as a highly acidic embryonic form (pI 5.16) is substituted by a basic larval form (pI 9.57). Thyroid hormones (THs), which play an important regulatory role in muscle development in flatfish and tetrapods, appear also to influence TnT gene expression in the sea bream. However, THs have a divergent action on different sea bream TnT genes and although the slow isoform (sTnT1) is TH-responsive, fTnT, sTnT2 and the itronless isoform (iTnT) are unaffected. The present results taken together with those published for flatfish seem to suggest differences may exist in the regulation of larval muscle development in teleosts.

Differential regulation of myofilament protein isoforms underlying the contractility changes in skeletal muscle unloading

Weight-bearing skeletal muscles change phenotype in response to unloading. Using the hindlimb suspension rat model, we investigated the regulation of myofilament protein isoforms in correlation to contractility. Four weeks of continuous hindlimb unloading produced progressive atrophy and contractility changes in soleus but not extensor digitorum longus muscle. The unloaded soleus muscle also had decreased fatigue resistance. Along with the decrease of myosin heavy chain isoform I and IIa and increase of IIb and IIx, coordinated regulation of thin filament regulatory protein isoforms were observed: gamma- and beta-tropomyosin decreased and alpha-tropomyosin increased, resulting in an alpha/beta ratio similar to that in normal fast twitch skeletal muscle; troponin I and troponin T (TnT) both showed decrease in the slow isoform and increases in the fast isoform. The TnT isoform switching began after 7 days of unloading and TnI isoform showed detectable changes at 14 days while other protein isoform changes were not significant until 28 days of treatment. Correlating to the early changes in contractility, especially the resistance to fatigue, the early response of TnT isoform regulation may play a unique role in the adaptation of skeletal muscle to unloading. When the fast TnT gene expression was upregulated in the unloaded soleus muscle, alternative RNA splicing switched to produce more high molecular weight acidic isoforms, reflecting a potential compensation for the decrease of slow TnT that is critical to skeletal muscle function. The results demonstrate that differential regulation of TnT isoforms is a sensitive mechanism in muscle adaptation to functional demands.

To breathe or not to breathe: the respiratory muscles and COPD

The respiratory muscles are unique among skeletal muscles in that they are required to work without sustained rest throughout life. It is not surprising, therefore, that the ventilatory pump has been the subject of intense investigation over the past 30 years, with particular interest in the ability

Chronic effects of low-frequency low-intensity electrical stimulation of stretched human muscle

Effects of low-frequency electrical stimulation, which is currently considered to be a possible countermeasure for long-duration spaceflights, with and without stretch were evaluated. Twelve young male volunteers were randomly distributed into two groups. In one group anterior thigh muscles—knee extensors of both legs were stimulated with frequency of 15 Hz for 4.5 wks, six times a week; each session was 6-h long. In the other group, electrical stimulation with the same parameters was applied to stretched knee extensors. Following stimulation the subjects exhibited an increase in fatigue resistance, and in the succinate dehydrogenase activity and a 10% gain in the percentage of muscle fibers with slow myosin heavy chain isoforms. In a stimulated group the peak voluntary strength went down significantly, the CSA of fast muscle fibers in m. quadriceps femoris became slightly less in size (10%). Electrical stimulation of the stretched muscles induced an insignificant decline in their strength and an increase of cross-sectional area of muscle fibers of both types. Thus chronic low-frequency electrical stimulation may be proposed as a candidate countermeasure against muscle strength and mass loss if it is combined with stretch.

Myosin heavy chain isoform transitions in canine skeletal muscles during postnatal growth

To gain a better understanding of the normal characteristics of developing canine muscles, myosin heavy chain (MHC) isoform expression was analysed in the axial and limb skeletal muscles of 18 young dogs whose ages ranged from the late prenatal stage to 6 months. We compared the results of immunohistochemistry using ten monoclonal antibodies, specific to different MHC isoforms, and enzyme-histochemical reactions, which demonstrate the activity of myofibrillar ATPase, succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH). In the skeletal muscles of fetuses and neonatal dogs the developmental isoforms MHC-emb and MHC-neo were prevalent. In all muscles the primary fibres, located centrally in each muscle fascicle, strongly expressed the slow isoform MHC-I. The adult fast isoform MHC-IIa was first noted in some of the secondary fibres on fetal day 55. During the first 10 days after birth, the expression of MHC-emb declined, as did that of MHC-neo during the second and third weeks. Correspondingly, the expression of MHC-IIa, and later, of MHC-I increased in the secondary fibres. Between the sixth week and second month the expression of MHC-IIx became prominent. The slow rhomboideus muscle exhibited an early expression of the slow isoform in the secondary fibres. Our results indicate that the timing of muscle maturation depends on its activity immediately following birth. The fastest developing muscle was the diaphragm, followed by the fast muscles. A pronounced changeover from developmental to adult isoforms was noted at 4-6 weeks of age, which coincides with the increased physical activity of puppies.

Protein kinase C theta co‐operates with calcineurin in the activation of slow muscle genes in cultured myogenic cells

Adult skeletal muscle fibers can be divided into fast and slow twitch subtypes on the basis of specific contractile and metabolic properties, and on distinctive patterns of muscle gene expression. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific genes (myoglobin (Mb), troponin I slow) in cultured skeletal muscle cells, as well as in transgenic mice, through the co-operation of peroxisome-proliferation-activator receptor gamma co-activator 1alpha (PGC1alpha) myocyte enhancer factor 2 (MEF2), and nuclear factor of activated T cells (NFAT) transcription factors. Specific protein kinase C isoforms have been shown to functionally co-operate with calcineurin in different cellular models. We investigated whether specific protein kinase C isoforms are involved in calcineurin-induced slow skeletal muscle gene expression. By pharmacological inhibition or exogenous expression of mutant forms, we show that protein kinase C theta (the protein kinase C isoform predominantly expressed in skeletal muscle) is required and co-operates with calcineurin in the activation of the Mb promoter, as well as in the induction of slow isoforms of myosin and troponin I expression, in cultured muscle cells. This co-operation acts primarily regulating MEF2 activity, as shown by using reporter gene expression driven by the Mb promoter mutated in the specific binding sites. MEF2 activity on the Mb promoter is known to be dependent on both PGC1alpha and inactivation of histone deacetylases (HDACs) activity. We show in this study that protein kinase C theta is required for, even though it does not co-operate in, PGC1alpha-dependent Mb activation. Importantly, protein kinase C theta regulates the HDAC5 nucleus/cytoplasm location. We conclude that protein kinase C theta ensures maximal activation of MEF2, by regulating both MEF2 transcriptional complex formation and HDACs nuclear export.