Myosin Heavy Chain Distribution Research Articles

Oxidative and glycolytic skeletal muscles deploy protective mechanisms to avoid atrophy under pathophysiological iron overload.

BackgroundIron excess has been proposed as an essential factor in skeletal muscle wasting. Studies have reported correlations between muscle iron accumulation and atrophy, either through ageing or by using experimental models of secondary iron overload. However, iron treatments performed in most of these studies induced an extra‐pathophysiological iron overload, more representative of intoxication or poisoning. The main objective of this study was to determine the impact of iron excess closer to pathophysiological conditions on structural and metabolic adaptations (i) in differentiated myotubes and (ii) in skeletal muscle exhibiting oxidative (i.e. the soleus) or glycolytic (i.e. the gastrocnemius) metabolic phenotypes.MethodsThe impact of iron excess was assessed in both in vitro and in vivo models. Murine differentiated myotubes were exposed to ferric ammonium citrate (FAC) (i.e. 10 and 50 μM) for the in vitro component. The in vivo model was achieved by a single iron dextran subcutaneous injection (1 g/kg) in mice. Four months after the injection, soleus and gastrocnemius muscles were harvested for analysis.Results In vitro, iron exposure caused dose‐dependent increases of iron storage protein ferritin (P < 0.01) and dose‐dependent decreases of mRNA TfR1 levels (P < 0.001), which support cellular adaptations to iron excess. Extra‐physiological iron treatment (50 μM FAC) promoted myotube atrophy (P = 0.018), whereas myotube size remained unchanged under pathophysiological treatment (10 μM FAC). FAC treatments, whatever the doses tested, did not affect the expression of proteolytic markers (i.e. NF‐κB, MurF1, and ubiquitinated proteins). In vivo, basal iron content and mRNA TfR1 levels were significantly higher in the soleus compared with the gastrocnemius (+130% and +127%; P < 0.001, respectively), supporting higher iron needs in oxidative skeletal muscle. Iron supplementation induced muscle iron accumulation in the soleus and gastrocnemius muscles (+79%, P < 0.001 and +34%, P = 0.002, respectively), but ferritin protein expression only increased in the gastrocnemius (+36%, P = 0.06). Despite iron accumulation, muscle weight, fibre diameter, and myosin heavy chain distribution remained unchanged in either skeletal muscle.ConclusionsTogether, these data support that under pathophysiological conditions, skeletal muscle can protect itself from the related deleterious effects of excess iron.

Organisational alteration of cardiac myofilament proteins by hyperglycaemia in mouse embryonic stem cell-derived cardiomyocytes.

The exposure of the developing foetal heart to hyperglycaemia in mothers with diabetes mellitus is a major risk factor for foetal cardiac complications that lead to heart failure. We studied the effects of hyperglycaemia on the layout of cardiac myofilament proteins in stem cell-derived cardiomyocytes and their possible underlying mechanisms. Mouse embryonic stem cells (mESCs) were differentiated into cardiac-like cells and cultured in media containing baseline- or high glucose concentrations. Cellular biomarkers were detected using Western blot analysis, immunocytochemistry, 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assay, and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. High glucose decreased the proportion of cardiac troponin T and α-actinin 2 positive mESCs as well as disrupted the α-actinin 2 striated pattern and the distribution of the cardiac myosin heavy chain α- and β isoforms. However, there was no alteration of the cellular EdU uptake nor the expression of the receptor of advanced glycation end-product (RAGE). High glucose also increased the presence of the oxidative stress marker nitrotyrosine as well as the number of TUNEL-stained nuclei in cardiac-like cells. Treatment with the antioxidant N-acetyl cysteine decreased the number of TUNEL-stained cells in high glucose and improved the α-actinin 2 striated pattern. Hyperglycaemia negatively impacted the expression and cellular organisation of cardiac myofilament proteins in mESC-derived cardiomyocytes through oxidative stress. The results add further insights into the pathophysiological mechanisms of cardiac contractile dysfunction in diabetic cardiac developmental disease.

Intermittent reloading does not prevent reduction in iron availability and hepcidin upregulation caused by hindlimb unloading.

What is the central question of this study? Could skeletal muscle be involved in microgravity-induced iron misdistribution by modulating expression of hepcidin, the master regulator of iron metabolism? What is the main finding and its importance? We demonstrate, in rats, that hepcidin upregulation is not a transient adaptation associated with early exposure to microgravity and that intermittent reloading does not limit microgravity-induced iron misdistribution despite having a beneficial effect on soleus muscle wasting. In humans, exposure to microgravity during spaceflight causes muscle atrophy, changes in iron storage and a reduction in iron availability. We previously observed that during 7 days of simulated microgravity in rats, hepcidin plays a key role in iron misdistribution, and we suggested that a crosstalk between skeletal muscle and liver could regulate hepcidin synthesis in this context. In the present study in rats, we investigated the medium-term effects of simulated microgravity on iron metabolism. We also tested whether intermittent reloading (IR) to target skeletal muscle atrophy limits iron misdistribution efficiently. For this purpose, Wistar rats underwent 14 days of hindlimb unloading (HU) combined or not combined with daily IR. At the end of this period, the serum iron concentration and transferrin saturation were significantly reduced, whereas hepatic hepcidin mRNA was upregulated. However, the main signalling pathways involved in hepcidin synthesis in the liver (BMP-small mothers against decapentaplegic (SMAD), interleukin-6-STAT3 and ERK1/2) were unaffected. Unlike what was observed after 7days of HU, the iron concentration in the spleen, liver and skeletal muscle was comparable between control animals and those that underwent HU or HU plus IR for 14 days. Despite its beneficial effect on soleus muscle atrophy and slow-to-fast myosin heavy chain distribution, IR did not significantly prevent a reduction in iron availability and hepcidin upregulation. Altogether, these results highlight that iron availability is durably reduced during longer exposure to simulated microgravity and that the related hepcidin upregulation is not a transient adaptation to these conditions. The results also suggest that skeletal muscle does not necessarily play a key role in the iron misdistribution that occurs during simulated microgravity.

Metabolic and Contractile Pathophysiology Following Volumetric Muscle Loss Injury

Volumetric muscle loss (VML) injury, due to abrupt and significant loss of tissue, exceeds the endogenous regenerative ability of the skeletal muscle, leading to long‐term functional impairment and disability. There is a well‐established physiologic relationship between skeletal muscle mass and metabolism, but the implications of VML injury on this relationship are widely unexplored. Recent investigations have identified oxidative and mitochondrial dysfunction associated with VML injury; however, the local metabolic myofiber response is not completely understood. We hypothesized that significant metabolic and contractile limitations would be observed in the remaining myofibers following VML injury, suggestive of motor unit reorganization. To evaluate the metabolic properties following VML, adult C57BL/6 male mice (n=16) underwent a sham procedure or multi‐muscle full thickness VML injury to the gastrocnemius, soleus, and plantaris muscles (21.1±0.7mg). Two months post‐VML, terminal histologic evaluation of capillarity, mitochondrial content, fibrotic deposition, and myosin heavy chain distribution was completed. Additionally, maximal contractile function and force‐frequency response of the posterior muscle compartment were evaluated. Maximum passive torque at 20 degrees dorsiflexion was significantly greater in the VML group (5.5±0.22 mN·m) compared to sham (2.3±0.13 mN·m). Maximum isometric torque was significantly less in VML (334±28 mN·m) compared to sham (557±66 mN·m). VML injury resulted in slower torque production and elevated twitch:tetani ratio, perhaps indicating a slower muscle fiber phenotype in the tissue remaining. Collectively, this work suggests that motor unit reorganization may alter muscle fiber phenotype following VML injury, likely limiting long‐term muscle functionality.Support or Funding InformationSupported by W81XWH‐18‐1‐0710 &amp; T32AR050938.

The Muscle Fiber Profiles, Mitochondrial Content, and Enzyme Activities of the Exceptionally Well-Trained Arm and Leg Muscles of Elite Cross-Country Skiers.

As one of the most physically demanding sports in the Olympic Games, cross-country skiing poses considerable challenges with respect to both force generation and endurance during the combined upper- and lower-body effort of varying intensity and duration. The isoforms of myosin in skeletal muscle have long been considered not only to define the contractile properties, but also to determine metabolic capacities. The current investigation was designed to explore the relationship between these isoforms and metabolic profiles in the arms (triceps brachii) and legs (vastus lateralis) as well as the range of training responses in the muscle fibers of elite cross-country skiers with equally and exceptionally well-trained upper and lower bodies. The proportion of myosin heavy chain (MHC)-1 was higher in the leg (58 ± 2% [34–69%]) than arm (40 ± 3% [24–57%]), although the mitochondrial volume percentages [8.6 ± 1.6 (leg) and 9.0 ± 2.0 (arm)], and average number of capillaries per fiber [5.8 ± 0.8 (leg) and 6.3 ± 0.3 (arm)] were the same. In these comparable highly trained leg and arm muscles, the maximal citrate synthase (CS) activity was the same. Still, 3-hydroxy-acyl-CoA-dehydrogenase (HAD) capacity was 52% higher (P < 0.05) in the leg compared to arm muscles, suggesting a relatively higher capacity for lipid oxidation in leg muscle, which cannot be explained by the different fiber type distributions. For both limbs combined, HAD activity was correlated with the content of MHC-1 (r2 = 0.32, P = 0.011), whereas CS activity was not. Thus, in these highly trained cross-country skiers capillarization of and mitochondrial volume in type 2 fiber can be at least as high as in type 1 fibers, indicating a divergence between fiber type pattern and aerobic metabolic capacity. The considerable variability in oxidative metabolism with similar MHC profiles provides a new perspective on exercise training. Furthermore, the clear differences between equally well-trained arm and leg muscles regarding HAD activity cannot be explained by training status or MHC distribution, thereby indicating an intrinsic metabolic difference between the upper and lower body. Moreover, trained type 1 and type 2A muscle fibers exhibited similar aerobic capacity regardless of whether they were located in an arm or leg muscle.

Chamber specific expression of Myosin heavy chain 7b in the heart of vertebrates

In extant vertebrates, myosin heavy chain (MyHC) 6 and 7 are the main isoforms of atrial and ventricular myocardium respectively, whereas MyHC7b has been proposed to be an ancient cardiac isoform only expressed during embryonic development in modern species. The MYHC7b DNA sequence has an intronic miRNA thought to be a regulator of slow‐twitch versus fast‐twitch muscle differentiation.The pan‐MyHC antibodies MF20 and A4.1025 are commonly used to highlight the myocardium in histomorphological studies. In preliminary immunohistochemical studies of the heart of the lesser spotted dogfish (Scyliorhinus canicula; Chondrichthyes), we have observed that while MF20 labels homogeneously all the myocardium, A4.1025 labels the inflow cardiac segments (sinus venosus and atrium) but not the outflow segments (ventricle and conus arteriosus). In order to interpret these results, we have performed western and slot blots from samples of dogfish and hamster (Mesocricetus auratus) hearts, HPLC‐ESI‐MS/MS from dogfish samples, and immunohistochemistry in hearts of representative species of vertebrates, namely elasmobranchs, polypteriforms, acipenseriforms, teleosts and mammals, using MF20 and A4.1025 antibodies.Western and slot blot results confirmed the specificity of MF20 and A4.1025 for MyHC in dogfish, as well as their differential reactivity against different myocardial segments. HPLC‐ESI‐MS/MS using protein databases from Callorhinchus milii (Chondrichthyes) and Chordata revealed the presence of MyHC6 and 7 in all the dogfish myocardial segments, and of MyHC7b only in the outflow segments. Immunohistochemistry showed that while MF20 signals were homogeneous in all the myocardial segments of all the species studied, A4.1025 signals were restricted to the inflow myocardial segments in elasmobranchs, homogeneous in teleosts and acipenseriforms, and low in the ventricle of polypteriforms.From our results it can be inferred that the A4.1025 antibody, as opposed to MF20, has a low affinity for MyHC7b, at least in the dogfish. In addition, we show that MyHC distribution in the cardiac chambers has changed during the evolution of gnathostomes. We raise the hypothesis that in early diverged groups of gnathostomes, MyHC6 and 7 were distributed in all the myocardial segments, whereas MyHC7b was specific for the outflow myocardium. In derived gnathostomes, MyHC6 and 7 became mainly restricted to the atrium and ventricle respectively, whereas MyHC7b disappeared from the heart, acquiring a regulatory role in cardiac and extracardiac myogenesis through its intronic miRNA.Support or Funding InformationThis study was supported by CGL2014‐52356‐P and FPU15/03209This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Segmental Distribution of Myosin Heavy Chain Isoforms Within Single Muscle Fibers.

Despite many studies looking at the distribution of myosin heavy chain (MHC) isoforms across a transverse section of muscle, knowledge of MHC distribution along the longitudinal axis of a single skeletal muscle fiber has been relatively overlooked. Immunocytochemistry was performed on serial sections of rat extensor digitorum longus (EDL) muscle to identify MHC types I, IIA, IIX, IIY, and IIB. Sixteen fascicles which contained a total of 362 fibers were randomly and systematically sampled from the three EDL muscles. All MHC type I and type II isoforms were expressed. Segmental expression occurred within a very limited segment. MHC isoform expression followed the accepted traditional order from I⇔IIA⇔IIX⇔IIB, however, in some samples expression of an isoform was circumvented from IIB to I or from I to IIB directly. Segmental distribution of MHC isoforms along a single muscle fiber may be because of the myonuclear domain. Anat Rec, 300:1636-1642, 2017. © 2017 Wiley Periodicals, Inc.

The functional significance of hamstrings composition: is it really a "fast" muscle group?

Hamstrings muscle fiber composition may be predominantly fast-twitch and could explain the high incidence of hamstrings strain injuries. However, hamstrings muscle composition invivo, and its influence on knee flexor muscle function, remains unknown. We investigated biceps femoris long head (BFlh) myosin heavy chain (MHC) composition from biopsy samples, and the association of hamstrings composition and hamstrings muscle volume (using MRI) with knee flexor maximal and explosive strength. Thirty-one young men performed maximal (concentric, eccentric, isometric) and explosive (isometric) contractions. BFlh exhibited a balanced MHC distribution [mean±SD (min-max); 47.1±9.1% (32.6-71.0%) MHC-I, 35.5±8.5% (21.5-60.0%) MHC-IIA, 17.4±9.1% (0.0-30.9%) MHC-IIX]. Muscle volume was correlated with knee flexor maximal strength at all velocities and contraction modes (r=0.62-0.76, P<0.01), but only associated with late phase explosive strength (time to 90Nm; r=-0.53, P<0.05). In contrast, BFlh muscle composition was not related to any maximal or explosive strength measure. BFlh MHC composition was not found to be "fast", and therefore composition does not appear to explain the high incidence of hamstrings strain injury. Hamstrings muscle volume explained 38-58% of the inter-individual differences in knee flexor maximum strength at a range of velocities and contraction modes, while BFlh muscle composition was not associated with maximal or explosive strength.

Short-term, high-fat diet accelerates disuse atrophy and protein degradation in a muscle-specific manner in mice.

BackgroundA short-term high-fat diet impairs mitochondrial function and the ability of skeletal muscle to respond to growth stimuli, but it is unknown whether such a diet alters the ability to respond to atrophy signals. The purpose of this study was to determine whether rapid weigh gain induced by a high-fat (HF) diet accelerates denervation-induced muscle atrophy.MethodsAdult, male mice (C57BL/6) were fed a control or HF (60 % calories as fat) diet for 3 weeks (3wHF). Sciatic nerve was sectioned unilaterally for the final 5 or 14 days of the diet. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated in vitro to determine rates of protein degradation and subsequently homogenized for determination of protein levels of LC3, ubiquitination, myosin heavy chain (MHC) distribution, and mitochondrial subunits.ResultsWhen mice were fed the 3wHF diet, whole-body fat mass more than doubled, but basal (innervated) muscle weights, rates of protein degradation, LC3 content, mitochondrial protein content, and myosin isoform distribution were not significantly different than with the control diet in either soleus or EDL. However in the 14 day denervated soleus, the 3wHF diet significantly augmented loss of mass, proteolysis rate, amount of the autophagosome marker LC3 II, and the amount of overall ubiquitination as compared to the control fed mice. On the contrary, the 3wHF diet had no significant effect in the EDL on amount of mass loss, proteolysis rate, LC3 levels, or ubiquitination. Fourteen days denervation also induced a loss of mitochondrial proteins in the soleus but not the EDL, regardless of the diet.ConclusionsTaken together, a short-term, high-fat diet augments denervation muscle atrophy by induction of protein degradation in the mitochondria-rich soleus but not in the glycolytic EDL. These findings suggest that the denervation-induced loss of mitochondria and HF diet-induced impairment of mitochondrial function may combine to promote skeletal muscle atrophy.

Structural and Functional Changes in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). The impact of HCM c/ssTnC mutations in skeletal muscle structure and function is unknown; therefore, we examined the effect of an HCM-associated c/ssTnC A8V mutation on myosin heavy chain (MHC) isoform expression, contractile properties of different skeletal muscles, and cardiac autonomic function in a homozygous knock-in mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in various muscles from 3-month old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE and the muscle/body weight ratios (M/BW) were recorded. Mechanics of contraction in isolated intact soleus muscle, running/endurance capacity and heart rate variability (HRV) were also measured. In comparison to the WT mice, MHC isoform switching was observed in diaphragm (increased MHCIIb, decreased MHCI) and soleus (decreased MHCIIa, increased MHCI) muscles of KI-TnC-A8V+/+ mice; while, the M/BW ratio was unchanged for all tested muscles. The force vs frequency relationship at 15Hz and the t1/2 of relaxation increased in the intact soleus muscle of KI-TnC A8V+/+ mice in comparison to the WT; whereas, no changes in maximal force, time-to-twitch peak, velocity and power output were observed. In endurance and HRV tests, KI-TnC-A8V+/+ mice had a lower running distance capacity and their global autonomic, cardiovagal, and baroreflex activities were significantly reduced compared to WT. In spite of the increase in type I fibers in soleus muscle of mice expressing c/ssTnC A8V, they display reduced endurance capacity. These findings suggest that the slow skeletal muscle is trying to compensate for the cardiac dysfunction caused by the c/ssTnC A8V+/+ mutation. Supported by NIH HL103840 (JRP).

Muscle Fibre Type Distribution of the Thoracolumbar and Hindlimb Regions of Horses: Relating Fibre Type and Functional Role

IntroductionThe muscle fibre type (MFT) of the deep epaxial musculature has not been determined in horses. The objective was to determine the MFT distribution in selected muscles of thoracolumbar and hindlimb region of horses. This included deep epaxial and hypaxial muscles that were hypothesised to have a postural stabilising role. A second objective was to examine differences in MFT distribution between Arabians and Quarter horses.MethodsMuscle biopsy samples (114) were obtained from 5 Quarter horses, 4 Arabians, and 2 Thoroughbreds. The myosin heavy chain distribution was determined by gel electrophoresis. Mann‐Whitney rank test was used to compare the proportional MFT content and differences between breeds.ResultsMm. sacrocaudalis dorsalis medialis and diaphragm had the highest proportion of MFT‐I, 84.1% (±18.4) and 72.4% (±12.5), respectively. The remaining deep epaxial muscles and the hypaxial m. psoas minor had approximately equal MFT‐I and II proportions. Mm. psoas major, iliocostalis, longissimus dorsi and the hind limb muscles contained mostly MFT‐IIX: from 50.5% (±12.8) to 67.9% (±7.3). Quarter horses had significantly more MFT‐IIX in m. psoas major 60.8% (±9.1) than Arabians 41.9% (±3.2). Arabians had significantly more MFT‐I fibres in m. longissimus dorsi 16.9% (±4.6) than Quarter horses 9.9% (±1.4) (P&lt;0.05).ConclusionsThe MFT distribution of the deep epaxial muscles, mm. psoas minor and diaphragm, suggest a postural stabilizing role in contrast to the fibre type proportions of the hindlimb muscles. The findings can direct diagnosis, prevention and treatment of muscular or neuromotor dysfunction in the thoracolumbar and hind limb area.Ethical Animal ResearchThe study was approved by the University of Queensland Animal Ethics Committee. Explicit owner informed consent for participation in this study is not stated. Sources of funding: none. Competing interests: none.

Muscle fibre type distribution of the thoracolumbar and hindlimb regions of horses: relating fibre type and functional role

BackgroundAlthough the majority of equine muscles have a mixed fibre type distribution indicative of diverse functional roles, the predominance of a fibre type can indicate the primary function of a muscle. The deep epaxial musculature has an important role in core spinal stability in humans, reflected as a predominantly muscle fibre type (MFT) I or postural fibre type. The fibre type of the deep epaxial musculature has not been determined in horses. The objective of the study was to determine the MFT distribution in selected muscles of thoracolumbar and hindlimb region of horses. This included deep epaxial and hypaxial muscles that were hypothesised to have a postural stabilising role. A second objective was to examine differences in MFT distribution between horses bred for endurance (Arabian) and sprinting (Quarter horse). Muscle biopsy samples were obtained from selected thoracolumbar and hind limb muscles of 5 Quarter horses, 4 Arabians, and 2 Thoroughbreds. The myosin heavy chain distribution was determined by gel electrophoresis. Mann–Whitney rank test was used to compare the proportional MFT and differences between breeds.ResultsMm. sacrocaudalis dorsalis medialis and diaphragm had the highest proportion of MFT-I. The remaining deep epaxial muscles and the hypaxial muscle m. psoas minor had approximately equal MFT I and II proportions. Mm. psoas major, iliocostalis, longissimus dorsi and the hind limb muscles contained mostly MFT-IIX. The fibre type distribution was similar between Arabians and Quarter horses, although Quarter horses had more MFT-IIX fibres in psoas major (P = 0.02) while Arabians had more MFT-I fibres in m. longissimus dorsi (P = 0.03).ConclusionsThe fibre type distribution of the deep epaxial muscles, mm psoas minor and diaphragm varied from approximately equal MFT-I and II proportions to predominantly MFT-I suggesting a postural stabilising role possibly important in core spinal stability. In contrast the fibre type proportions of mm psoas major, iliocostalis, longissimus dorsi and the hind limb muscles were mainly MFT-II suggesting a locomotory role. Knowledge of fibre type distribution in such a clinically important area can direct diagnosis, prevention and treatment of muscular or neuromotor dysfunction.

Myosin Heavy Chain Isoform Switching in Skeletal Muscles in an A8V-Troponin C Hypertrophic Cardiomyopathy Knock-In Mouse Model

Missense mutations in the TNNC1 gene encoding cardiac and slow skeletal troponin C (c/ssTnC) are associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM). Since the impact of HCM c/ssTnC mutations during skeletal muscle development and function is unknown, we addressed whether changes in the myosin heavy chain (MHC) isoforms can occur outside of the heart. We hypothesized that the ratio of MHC I to MHC II in various skeletal muscles change due to an HCM-associated c/ssTnC A8V mutation in a knock-in homozygous mouse (KI-TnC-A8V+/+). The distribution of MHC I/II isoforms in diaphragm (D), soleus (S), tibialis anterior (TA), extensor digitorum longus (EDL) and quadriceps (Q) isolated from 3- and 9-month (mo) old male WT and KI-TnC-A8V+/+ mice were analyzed by glycerol SDS-PAGE. The muscle/body weight ratio (M/BW) and running capacity were also measured. At 3 mo, MHC isoform switching was observed in D (increased MHCIIb), S (decreased MHCIIa, increased MHCI) and EDL (increased MHCIIa/x, decreased MHCIIb) of KI-TnC-A8V+/+ compared to WT. TA M/BW is increased in 3- and 9-mo KI-TnC-A8V+/+ mice compared to WT at the same age. Pairwise comparison of M/BW from 3- vs 9-mo KI-TnC-A8V+/+ mice showed a significant decrease for TA and Q and an increase for EDL, and no change for any WT muscles. In running tests, KI-TnC-A8V+/+ mice had a lower capacity to run distances compared to WT (281 vs 413 meters). Thus, changes in MHC isoforms in the skeletal muscles of mice expressing c/ssTnC A8V are associated with reduced exercise endurance, suggesting that skeletal muscle remodeling may contribute to the development of HCM phenotype. Future studies will examine MHC isoforms at 9 mo and Ca2+-activated myofibrilar Mg2+-ATPase rates from skeletal muscles in KI-TnC-A8V+/+ mice.

Cardiac myosin heavy chains in mice treated with NG-nitro-l-arginine methyl ester and thyroxine

The aim of this study was to evaluate the effect of NG-nitro-l-arginine methyl ester and thyroxine on the distribution of cardiac myosin heavy chains in mice. Myocardial hypertrophy was induced in mice by intraperitoneal injection with l-thyroxine and oral administration of NG-nitro-l-arginine methyl ester (l-NAME). Mice were randomly allocated into five groups: control, high and low doses of thyroxine and L-NAME. Heart weight divided by body weight was calculated and used as indicator for cardiac hypertrophy. Myosin heavy chains (MHCs) were separated using 4 % polyacrylamide gel electrophoresis. Hypertrophy was induced in mice treated with thyroxine and a high dose of l-NAME and was accompanied by a shift toward α-MHC in thyroxine-treated mice and β-MHC in l-NAME-treated mice. There was no difference with respect to MHC between a low dose of l-NAME and a high dose; however, low doses of l-NAME did not result in cardiac hypertrophy. In conclusion, l-NAME treatment changes the MHC distribution from α- toward β-MHC and this transition in the MHCs occurs before heart hypertrophy. Further investigations are needed to determine whether l-NAME treatment causes this transition via a direct or indirect effect on the heart muscles.

The characteristics of myosin heavy chain-based fiber types in porcine longissimus dorsi muscle

The aim of this study is to type fibers from porcine longissimus dorsi (LD) muscles according to their distribution of myosin heavy chain (MHC) isoforms as well as to investigate fiber characteristics. Four pure types, including types I, IIA, IIX, and IIB were labeled and two hybrid fiber types were subdivided into type IIAX and IIXB by immunohistochemistry using four monoclonal antibodies. Porcine LD muscles were found to have 92.79 (number) and 92.10% (area) of pure type composition, while the composition of hybrid fibers was 8.22 (number) and 9.71% (area). The activities of myosin ATPase increased in the following order: type I, IIAX, IIA=IIX=IIB and IIXB. The succinate dehydrogenase and glycerol-3-phosphate dehydrogenase activities were higher in fiber types I and IIB, respectively. The characteristics of hybrid fibers were observed that their characteristics did not lie between the properties for their respective pure phenotypes.

Effect of hypoxia exposure on the phenotypic adaptation in remodelling skeletal muscle submitted to functional overload

To determine whether hypoxia influences the phenotypic adaptation of skeletal muscle induced by mechanical overload. Plantaris muscles of female rats were submitted to mechanical overload following synergist ablation. After 3 days of overload, rats were exposed to either hypobaric hypoxia (equivalent to 5500 m) or normoxia. Muscles were collected after 5, 12 and 56 days of overload (i.e. after 3, 9 and 53 days of hypoxia). We determined the myosin heavy chain (MHC) distribution, mRNA levels of myocyte-enriched calcineurin-integrating protein 1 (MCIP1) to indirectly assess calcineurin activity, the changes in oxidative capacity from the activities of citrate synthase (CS) and cytochrome c oxidase (COX), and the expression of regulators involved in mitochondrial biogenesis (Pgc-1α, NRF1 and Tfam) and degradation (BNIP-3). Hypoxia did not alter the fast-to-slow MHC shift and the increase in calcineurin activity induced by overload; it only transiently slowed down the overload-induced transition in MHC isoforms. Hypoxia similarly decreased CS and COX activities in overloaded and control muscles. Nuclear respiratory factor 1 (NRF1) and transcription factor A (Tfam) mRNA and BNIP-3 protein were not influenced by hypoxia in overloaded muscles, whereas Pgc-1α mRNA and protein contents did not correlate with changes in oxidative capacity. Hypoxia is not a critical stimulus to modulate the fast-to-slow MHC transition associated with overload. Thus, the impairment of the fast-to-slow fibre shift often observed during post-natal development in hypoxia could be explained by the lower voluntary locomotor activity associated with hypoxia. Hypoxia alters mitochondrial oxidative capacity, but this adaptive response is similar in overloaded and control muscles.

Adaptations in physiological properties of rat motor units following 5 weeks of whole-body vibration

The purpose of the study was to determine the effects of 5-week whole-body vibration (WBV) on contractile parameters and force-frequency relationship of functionally isolated motor units of the rat medial gastrocnemius muscle: fast fatigable (FF), fast fatigue-resistant (FR), and slow (S). Moreover, myosin heavy chain isoform content was quantified. Following WBV, the maximum tetanic force of FF units was increased by ∼25%. The twitch half-relaxation time in all types of motor units and the twitch contraction time in FR units were shortened. The twitch-to-tetanus force ratio was decreased and the force-frequency curves were shifted rightwards in S and FR units. Myosin heavy chain distribution was not changed. These findings suggest modifications of the excitation-contraction coupling towards shortening of a twitch contraction. The observed increase in force of FF units may contribute to gains in muscle dynamic strength reported following WBV treatment.

Training‐induced acceleration of O2 uptake on‐kinetics precedes muscle mitochondrial biogenesis in humans

The effects of 5 weeks of moderate-intensity endurance training on pulmonary oxygen uptake kinetics (V(O(2)) on-kinetics) were studied in 15 healthy men (mean ± SD: age 22.7 ± 1.8 years, body weight 76.4 ± 8.9 kg and maximal V(O(2)) 46.0 ± 3.7 ml kg(-1) min(-1)). Training caused a significant acceleration (P = 0.003) of V(O(2)) on-kinetics during moderate-intensity cycling (time constant of the 'primary' component 30.0 ± 6.6 versus 22.8 ± 5.6 s before and after training, respectively) and a significant decrease (P = 0.04) in the amplitude of the primary component (837 ± 351 versus 801 ± 330 ml min(-1)). No changes in myosin heavy chain distribution, muscle fibre capillarization, level of peroxisome proliferator-activated receptor γ coactivator 1α and other markers of mitochondrial biogenesis (mitochondrial DNA copy number, cytochrome c and cytochrome oxidase subunit I contents) in the vastus lateralis were found after training. A significant downregulation in the content of the sarcoplasmic reticulum ATPase 2 (SERCA2; P = 0.03) and a tendency towards a decrease in SERCA1 (P = 0.055) was found after training. The decrease in SERCA1 was positively correlated (P = 0.05) with the training-induced decrease in the gain of the V(O(2)) on-kinetics (ΔV(O(2)) at steady state/Δpower output). In the early stage of training, the acceleration in V(O(2)) on-kinetics during moderate-intensity cycling can occur without enhanced mitochondrial biogenesis or changes in muscle myosin heavy chain distribution and in muscle fibre capillarization. The training-induced decrease of the O(2) cost of cycling could be caused by the downregulation of SERCA pumps.

Variability in Muscle Adaptation to Electrical Stimulation

The aims were to investigate the plasticity of the myosin heavy chain (MHC) phenotype following neuromuscular electrical stimulation (NMES) and to assess the correlation between MHC isoform distribution and muscle fibre conduction velocity (MFCV).14 men were subjected to 24 sessions of quadriceps NMES. Needle biopsies were taken from the dominant vastus lateralis and neuromuscular tests were performed on the dominant thigh before and after training. NMES significantly increased the quadriceps maximal force by 14.4±19.7% (P=0.02), vastus lateralis thickness by 10.7±8.6% (P=0.01), vastus lateralis MFCV by 11.1±3.5% (P<0.001), vastus medialis MFCV by 8.4±1.8% (P<0.001). The whole spectrum of possible MHC isoform adaptations to training was observed: fast-to-slow transition (4 subjects), bi-directional transformation from MHC-1 and MHC-2X isoforms toward MHC-2A isoform (7 subjects), shift toward MHC-2X (2 subjects), no MHC distribution change (1 subject). No significant correlation was observed between MHC-2 relative content and vastus lateralis MFCV (pre-training: R2=0.04, P=0.46; post-training: R2=0.02, P=0.67). NMES elicited distinct adaptations in the MHC composition and increased force, muscle thickness, and MFCV. The MHC isoform distribution did not correlate with MFCV, thus implying that the proportion of different fibre types cannot be estimated from this electrophysiological variable.

Insulin and Fiber Type in the Offspring of T2DM Subjects with Resistance Training and Detraining

Effects of resistance training and detraining on glucose and insulin responses to an oral glucose load, muscle fiber type, and muscular performance in the offspring of those with type 2 diabetes (familial insulin resistant (FIR)) were investigated. Six FIR participants and 10 controls (C) completed 9 wk of resistance training and 9 wk of detraining. Measures of strength and power, an oral glucose tolerance test, and a muscle biopsy to determine myosin heavy chain (MHC) fiber composition were taken at baseline (T1), after training (T2), and after detraining (T3). Three-repetition maximum increased (P ≤ 0.001) similarly in both groups in all strength measures, e.g., leg press (FIR T1, T2: 121 ± 34 kg, 186 ± 50 kg; C T1, T2: 137 ± 42 kg, 206 ± 64 kg, respectively (means ± SD)). Wingate peak power increased (FIR T1, T2: 505 ± 137 W, 523 ± 143 W; C T1, T2: 636 ± 211 W, 672 ± 223 W, respectively; P ≤ 0.005 (means ± SD)). Training reduced insulin area under the curve more (P = 0.050) in FIR (T1, T2: 1219 ± 734 pmol·L, 837 ± 284 pmol·L, respectively (means ± SD)) than that in C (T1, T2: 647 ± 268 pmol·L, 635 ± 258 pmol·L, respectively (means ± SD)). MHC distribution did not change with training. Strength (three-repetition maximum measures) decreased with detraining (P ≤ 0.001) although Wingate power did not. Detraining increased insulin area under the curve (P = 0.018) in FIR (T2, T3: 837 ± 285 pmol·L, 1040 ± 194 pmol·L, respectively (means ± SD)) but not in C (T2, T3: 635 ± 258 pmol·L, 625 ± 213 pmol·L, respectively (means ± SD)). MHC IIX fibers increased with detraining (P = 0.026). FIR appears to have exaggerated responses to resistance training and detraining, with a greater reduction in insulin release with glucose ingestion after training and increase when training ceases. Resistance training has a significant effect on insulin responses and may reduce future risk of type 2 diabetes mellitus among FIR.