Equine Skeletal Muscle Research Articles

Optimalization of preparation of apocytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3–6 (10 mM KH2PO4, pH 3–6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here.

Effects of dexamethasone administration on insulin resistance and components of insulin signaling and glucose metabolism in equine skeletal muscle

To determine the effects of dexamethasone treatment on selected components of insulin signaling and glucose metabolism in skeletal muscle obtained from horses before and after administration of a euglycemic-hyperinsulinemic clamp (EHC). 6 adult Standardbreds. In a balanced crossover study, horses received either dexamethasone (0.08 mg/kg, IV, q 48 h) or an equivalent volume of saline (0.9% NaCl) solution, IV, for 21 days. A 2-hour EHC was administered for measurement of insulin sensitivity 1 day after treatment. Muscle biopsy specimens obtained before and after the EHC were analyzed for glucose transporter 4, protein kinase B (PKB), glycogen synthase kinase (GSK)-3alpha/beta protein abundance and phosphorylation state (PKB Ser(473) and GSK-3alpha/beta Ser(21/9)), glycogen synthase and hexokinase enzyme activities, and muscle glycogen concentration. Dexamethasone treatment resulted in resting hyperinsulinemia and a significant decrease (70%) in glucose infusion rate during the EHC. In the dexamethasone group, increased hexokinase activity, abrogation of the insulin-stimulated increase in glycogen synthase fractional velocity, and decreased phosphorylation of GSK-3alpha Ser(21) and GSK-3B Ser(9) were detected, but there was no effect of dexamethasone treatment on glucose transporter 4 content and glycogen concentration or on PKB abundance and phosphorylation state. In horses, 21 days of dexamethasone treatment resulted in substantial insulin resistance and impaired GSK-3 phosphorylation in skeletal muscle, which may have contributed to the decreased glycogen synthase activity seen after insulin stimulation.

Muscle energetics in exercising horses

AbstractAn optimally functional musculoskeletal system is crucial for athletic performance and even minor perturbations can limit athletic ability. The introduction of the muscle biopsy technique in the 1970s created a window of opportunity to examine the form and function of equine skeletal muscle. Muscle histochemical and biochemical analyses have allowed characterization of the properties of equine muscle fibres and their influence on, and adaptation to, physical exertion. Analyses of exercise responses during standardized treadmill exercise and field studies have illustrated the role of cellular energetics in determining athletic suitability for specific disciplines, mechanisms of fatigue, adaptations to training and the affect of diet on metabolic responses. This article provides a review of the tools available to study muscle energetics in the horse, discusses the muscular metabolic pathways and summarizes the energetics of exercise.

Characterization of the equine glycogen debranching enzyme gene ( AGL): Genomic and cDNA structure, localization, polymorphism and expression

Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting in the glycogen degradation pathway. In humans, the AGL activity deficiency causes a type III glycogen storage disease (Cori–Forbes disease). One particularity of AGL gene expression lies in the multiple alternative splicing in its 5′ region. The AGL gene was localized on ECA5q14-q15. The sequence of the equine cDNA was determined to be 7.5 kb in length with an open reading frame of 4602 bp. The gene is 69 kb long and contains 35 exons. The equine AGL gene has an ubiquitous expression and presents five tissue-dependent cDNA variants arising from alternative splicing of the first exons. The equine skeletal muscle and heart contain four out of six variants previously described in humans and the equine liver express three of these four human variants. We identified a new alternative splicing variant expressed in equine skeletal and heart muscles. All these mRNA variants most probably encode only two different protein isoforms of 1533 and 1377 amino-acids. Four SNPs were detected in the mRNA. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species. The disposition of the transcription factor biding sites does not correlate to the transcription start sites of tissue-specific variants.

A Scientific Background for Skeletal Muscle Conditioning in Equine Practice

The main goal of any conditioning programme in athletic horses is to improve performance by inducing physiological changes within the animal's body. Equine skeletal muscles have a considerable potential to adapt during training and these adaptations have important physiological implications that influence stamina, strength and speed. Although there is an extensive specialized literature in this regard, scientific based muscle conditioning methods have not been introduced sufficiently in the equine sport practice. After a brief synopsis of both equine muscle exercise physiology and muscular adaptations to training, including their physiological significance, this review focuses on specific training programmes that induce muscular adaptations in athletic horses. The article addresses the following principal question: what kind of stimuli for what kind of muscular adaptations? The experimental data are discussed separately for racehorses (thoroughbreds, trotters and endurance horses) and sport horses (dressage, show jumpers and carriage). Finally, published results about the influence of relevant training parameters (such as intensity, duration and type of exercise) on muscular responses are discussed, as well as those concerning overtraining and detraining. The article closes with some concluding remarks on importance of their application in practice.

Can Apomyoglobin Form a Complex with a Spherical Ligand? Interactions Between Apomyoglobin and [C60] Fullerene Derivative

The preparation and characterization of the stable equine skeletal muscle apomyoglobin and eee-isomer of tris-malonic acid [C60] fullerene complex is reported. For this new bio-nanomaterial preparation, a procedure of complexation-during-protein-refolding was used and the obtained compound sustained separation by gel-filtration and ion-exchange chromatography. The apomyoglobin-tris-malonic acid [C60] fullerene complex was characterized by UV-vis spectroscopy, steady state fluorescence, time-resolved fluorescence, and circular dichroism spectroscopies. Important information provided by this study, regarding the stability and properties of new material, may lead to a better understanding of the apomyoglobin protein binding characteristics, as well as to development of novel antioxidant and photodynamic therapeutic agents and components for bioelectronic devices.

Sequencing of cDNA and proximal promoter of equine hexokinase II gene

In order to investigate the utilization of glucose in equine skeletal muscle, we determined the coding and proximal promoter sequences of the hexokinase type II (HKII) gene in thoroughbred horse, Grevy's zebra and Hartmann's mountain zebra. The deduced amino acid sequence of thoroughbred horse HKII showed 100, 100, 94.4, 92.7 and 92.6% identities with Grevy's zebra, Hartmann's mountain zebra, human, mouse and rat HKIIs, respectively. In equine HKIIs, specific amino acid substitutions, Ile 159 and Arg 610, were found in the potential binding site for glucose. In addition, the nucleotide sequence of the equine proximal promoter has a number of conserved putative binding sites for transcription factor.

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Effects of prior exercise on components of insulin signalling in equine skeletal muscle

Exercise is an important stimulus for the regulation of numerous metabolic functions in muscle, but there has been little investigation of the mechanism of exercise-enhanced glucose utilisation. To examine the effects of exercise on the signalling components Akt and glycogen synthase kinase-3 (GSK3) involved in insulin regulation of glycogen synthase (GS) activity. It was hypothesised that hyperinsulinaemia and prior exercise would alter the extent of Akt phosphorylation and GSK3beta deactivation and increase insulin stimulation of GS activity in skeletal muscle. Eight Standardbred horses completed a euglycaemic-hyperinsulinaemic clamp (EHC) either without prior exercise (NonEX) or at 0.5, 4 or 24 h post exercise (0.5 PostEX, 4 PostEX, and 24 PostEX, respectively). Muscle samples for determination of Akt and GSK3 serine phosphorylation, and GS fractional velocity (GS(FV)) were collected 5 min before (Pre-INS) and at the end (Post-INS) of each EHC (pre- and post insulin stimulation). Protein was separated by SDS-PAGE on polyacrylamide gels, transferred to nitrocellulose membranes and incubated with antibodies against phospho-Akt Ser473 and GSK3alpha/beta Ser21/9. GS(FV) was increased (P<0.05) 2-fold at 0.5 PostEX, 4 PostEX and 24 PostEX when compared to NonEX in Pre-INS and in Post-INS was increased 33 and 28% in NonEX and at 24PostEX (P<0.05), respectively, when compared to Pre-INS in these trials. Phospho-Akt Ser473 was increased at 0.5 PostEX only (P<0.05) in Pre-INS but increased in all trials in Post-INS. Phospho-GSK3alpha (Ser21) was also greater (P<0.05) at 0.5 PostEX in Pre-INS than in NonEX and 24 PostEX and in Post-INS increased (P<0.05) in NonEX and at 24 PostEX but not at 0.5 PostEX or 4 PostEX post exercise. There was no effect of time or treatment on GSK3, (Ser9) phosphorylation or on total GSK3. Elevated plasma insulin and prior exercise were associated with Akt activation and GSK3alpha deactivation (0.5 PostEX) indicating that an effect of exercise is evident in the early post exercise period and supporting a physiological role for GSK3alpha in the regulation of GS activity. The changes are consistent with insulin signalling downstream from these molecules.

Evaluation of Multiprotein Immunoaffinity Subtraction for Plasma Proteomics and Candidate Biomarker Discovery Using Mass Spectrometry

Strategies for removal of high abundance proteins have been increasingly utilized in proteomic studies of serum/plasma and other body fluids to enhance the detection of low abundance proteins and achieve broader proteome coverage; however, both the reproducibility and specificity of the high abundance protein depletion process still represent common concerns. Here we report a detailed evaluation of immunoaffinity subtraction performed applying the ProteomeLab IgY-12 system that is commonly used in human serum/plasma proteome characterization in combination with high resolution LC-MS/MS. Plasma samples were repeatedly processed using this approach, and the resulting flow-through fractions and bound fractions were individually analyzed for comparison. The removal of target proteins by the immunoaffinity subtraction system and the overall process was highly reproducible. Non-target proteins, including one spiked protein standard (rabbit glyceraldehyde-3-phosphate dehydrogenase), were also observed to bind to the column at different levels but also in a reproducible manner. The results suggest that multiprotein immunoaffinity subtraction systems can be readily integrated into quantitative strategies to enhance detection of low abundance proteins in biomarker discovery studies.

Effect of exercise on activation of the p38 mitogen-activated protein kinase pathway, c-Jun NH2 terminal kinase, and heat shock protein 27 in equine skeletal muscle

To investigate the effects of exercise on activation of mitogen-activated protein kinase (MAPK) signaling proteins in horses. 6 young trained Standardbred geldings. Horses performed a 20-minute bout of exercise on a treadmill at 80% of maximal heart rate. Muscle biopsy specimens were obtained from the vastus lateralis and pectoralis descendens muscles before and after exercise. Amount of expression and intracellular location of phosphospecific MAPK pathway intermediates were determined by use of western blotting and immunofluorescence staining. Exercise resulted in a significant increase in phosphorylation of p38 pathway intermediates, c-Jun NH2 terminal kinase (JNK), and heat shock protein 27 (HSP27) in the vastus lateralis muscle, whereas no significant changes were found in phosphorylation of extracellular regulated kinase. In the pectoralis descendens muscle, phosphorylation of p38 and HSP27 was significantly increased after exercise. Immunohistochemical analysis revealed fiber-type- specific locations of phosphorylated JNK in type 2a/b intermediate and 2b fibers and phosphorylated p38 in type 1 fibers. Phosphorylated HSP27 was strongly increased after exercise in type 1 and 2a fibers. The p38 pathway and JNK are activated in the vastus lateralis muscle after a single 20-minute bout of submaximal exercise in trained horses. Phosphorylation of HSP27 as detected in the study reported here is most likely induced through the p38 signaling pathway.

Evaluation of developmental changes in the coexpression of myosin heavy chains and metabolic properties of equine skeletal muscle fibers

To determine the growth-related changes in metabolic and anatomic properties in equine muscle fiber type, including hybrid fibers identified with immunohistochemical analysis. 24 2-, 6-, 12-, and 24-month-old female Thoroughbreds. Samples were obtained from the gluteus medius muscle of all horses. Expression of myosin heavy chain (MHC) isoforms MHC-I, -IIa, -IIb, and -IIx in each muscle fiber was detected by use of 4 primary monoclonal antibodies: BA-D5, SC-71, BF-F3, and BF-35, respectively. Five muscle fiber types (types I, I/IIA, IIA, IIA/IIX, and IIX) were immunohistochemically identified. The area and activity of succinic dehydrogenase (SDH) in each fiber type were determined by use of quantitative histochemical staining and image analysis. Although the proportion of type I and IIX fibers did not change with age, the proportion of type IIA and IIA/IIX fibers significantly increased and decreased, respectively, from 2 months to 24 months of age. The increase in proportion of type IIA fibers with growth may have been attributable to muscle fiber-type transition from type IIA/IIX fibers but not from type IIX fibers. Values for SDH activity and fiber area in hybrid fiber types were intermediate to those for their respective pure phenotypes. Hybrid fibers have an important role for determining the proportion of muscle fiber type in horses < 24 months old, and the metabolic and anatomic properties of the hybrid fibers are well coordinated, as in mature horses.

Sarcoplasmic masses in equine skeletal muscle

Sarcoplasmic masses in humans have been associated with various myopathies, although the significance remains elusive. Similar structures have also been observed in equine muscle. The aim of this study was to determine the frequency of such structures in normal and abnormal equine muscle, and to characterize these structures using histological, histochemical, immunohistochemical, ultrastructural, and morphometric analyses. The histological and histochemical appearance was similar to that of human sarcoplasmic masses with a central or subsarcolemmal distribution. Of interest was a predilection for the gluteus medius muscle in younger horses and type 2B fibers. Ultrastructurally they contained disorganized myofibrils and other cellular components that were not membrane bound and were present in both normal and abnormal equine muscle without a specific disease association, suggesting they are a non-pathological degenerative structure. The relatively frequent occurrence of sarcoplasmic masses in horses may make this species a good model for studying the pathogenesis of these structures.

Age-related changes in metabolic properties of equine skeletal muscle associated with muscle plasticity

The purpose of the present study was to determine the age-related changes in myosin heavy chain (MHC) composition and muscle oxidative and glycolytic capacity in 18 horses ranging in age from two to 30 years. Muscle samples were collected by excisional biopsy of the semimebranosus muscle. MHC expression and the key enzymatic activities were measured. There was no significant correlation between horse age and the proportions of type-IIA and type-IIX MHC isoforms. The percentage of type-I MHC isoforms decreased with advancing age. Muscle citrate synthase activity decreased, whereas lactate dehydrogenase activity increased with increasing age. Muscle 3-OH acyl CoA dehydrogenase activity did not change with ageing. The results suggest that, similar to humans, the oxidative capacity of equine skeletal muscle decreases with age. The age-related changes in muscle metabolic properties appear to be consistent with an age-related transition in MHC isoforms of equine skeletal muscle that shifts toward more glycolytic isoforms with age.

Light-activated transfer of nitric oxide from a porous material.

A light-dark-light-dark sequence was used to demonstrate that only NO is released from a new mesoporous material that contains immobilized Ru(NO) sites. No release is observed during the dark phases (shown schematically). Since this release is regulated by light, the controlled transfer of NO to cobalt(II) tetraphenylphorphyrin as well as equine skeletal muscle myoglobin is possible.

Age-related changes in metabolic properties of equine skeletal muscle associated with muscle plasticity

The purpose of the present study was to determine the age-related changes in myosin heavy chain (MHC) composition and muscle oxidative and glycolytic capacity in 18 horses ranging in age from two to 30 years. Muscle samples were collected by excisional biopsy of the semimebranosus muscle. MHC expression and the key enzymatic activities were measured. There was no significant correlation between horse age and the proportions of type-IIA and type-IIX MHC isoforms. The percentage of type-I MHC isoforms decreased with advancing age. Muscle citrate synthase activity decreased, whereas lactate dehydrogenase activity increased with increasing age. Muscle 3-OH acyl CoA dehydrogenase activity did not change with ageing. The results suggest that, similar to humans, the oxidative capacity of equine skeletal muscle decreases with age. The age-related changes in muscle metabolic properties appear to be consistent with an age-related transition in MHC isoforms of equine skeletal muscle that shifts toward more glycolytic isoforms with age.

Preliminary evidence for dormant clostridial spores in equine skeletal muscle.

Equine Veterinary JournalVolume 35, Issue 5 p. 514-516 Preliminary evidence for dormant clostridial spores in equine skeletal muscle M. VENGUST, Corresponding Author M. VENGUST Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaDepartment of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorL. G. ARROYO, L. G. ARROYO Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorJ. S. WEESE, J. S. WEESE Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorJ. D. BAIRD, J. D. BAIRD Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this author M. VENGUST, Corresponding Author M. VENGUST Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaDepartment of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorL. G. ARROYO, L. G. ARROYO Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorJ. S. WEESE, J. S. WEESE Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this authorJ. D. BAIRD, J. D. BAIRD Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, CanadaSearch for more papers by this author First published: 05 January 2010 https://doi.org/10.2746/042516403775600569Citations: 20AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume35, Issue5July 2003Pages 514-516 RelatedInformation

Molecular Cloning of Equine (Thoroughbred) Myostatin cDNA and Detection of Myostatin Precursor Proteins in the Serum.

Growth/Differentiation Factor 8, or GDF-8, is a member of the TGF-β family and is expressed specifically in myogenic precursor cells of the myotome during development as well as in adult axial and paraxial muscles of the mouse. It acts as a negative regulator of skeletal muscle mass, and is known as myostatin. As a part of our studies to elucidate mechanisms underlying the growth of skeletal muscle mass due to physical training in mammals including horses and humans, we isolated and cloned myostatin cDNA from equine (Thoroughbred) skeletal muscles by an RT-PCR method. The base sequence of the entire coding region of equine myostatin cDNA (DDBJ accession no. AB033541) exhibited a high degree of homology to those of other species so far reported. In the deduced amino acid sequence, 4 amino acid positions were unique to the equine myostatin, i. e., Val15 instead of Met, Leu18 instead of Val or Ala, Ala201 instead of Thr, Arg244 instead of Gly or Glu in other species. Western blot analysis of the equine skeletal muscles using anti-myostatin antibody, yielded 3 immunoreactive bands, i. e., 26, 45 and 50 kDa. The band at approximately 26 kDa probably represents mature myostatin and another at approximately 50 kDa corresponds in size to the unprocessed precursor myostatin proteins reported in the literature. Western blot analysis of equine sera of 2 independent breeds, Thoroughbred and Kiso-uma, revealed the possible presence of precursor myostatin proteins at relatively high levels. The molecular identity and the biological significance of serum myostatin immunoreactivities remain to be clarified.

Co-ordinated expression of contractile and non-contractile features of control equine muscle fibre types characterised by immunostaining of myosin heavy chains.

Combined methodologies of immunohistochemistry, histochemistry and photometric image analysis were applied: (1) to characterise control equine skeletal muscle fibres according to their myosin heavy chain (MyHC) composition and (2) to determine on a fibre-to-fibre basis the correlation between contractile [i.e. MyHC(s), myofibrillar ATPase (mATPase) and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) isoforms], metabolic [i.e. succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (GPD) activities, glycogen and phospholamban (PLB) contents], and morphological [i.e. cross-sectional area (CSA), capillary and nuclear densities] features of individual myofibres. An accurate delineation of MyHC-based fibre types was obtained with the immunohistochemical method developed. This protocol showed a high sensitivity and objectivity to delineate hybrid fibres with overwhelming dominance of one MyHC isoform and, furthermore, it allowed a semiquantitative delineation of fast hybrid fibres according to the predominant MyHC isoform expressed. The phenotypic differences in contractile, metabolic and morphological properties seen between fibre types were related to MyHC content. Slow fibres had the lowest mATPase activity (related to shortening velocity), the highest SDH activity (oxidative capacity), the lowest GPD activity (glycolytic metabolism) and glycogen content, the smallest CSA, the greatest capillary and nuclear densities, and expressed slow SERCA isoform and PLB, but not the fast SERCA isoform. The reverse pattern was true for pure IID/X fibres, and type IIA fibres had intermediate properties. Hybrid IIAD/X fibres had mean values intermediate to those of their respective pure phenotypes. Discrimination of fibres according to their MyHC content was possible on the basis of their contractile and non-contractile profiles. These intrafibre interdependencies suggest that, even when controlled by different mechanisms, myofibres of control horses exhibit a high degree of co-ordination in their physiological, biochemical and anatomical features.

Early and long-term changes of equine skeletal muscle in response to endurance training and detraining.

Twenty-four 4-year-old Andalusian (Spanish breed) stallions were used to examine the plasticity of myosin heavy chain (MHC) phenotype and the metabolic profile in horse skeletal muscle with long-term endurance-exercise training and detraining. Sixteen horses underwent a training programme based on aerobic exercises for 8 months. Afterwards, they were kept in paddocks for 3 months. The remaining eight horses were used as controls. Three gluteus medius muscle biopsy samples were removed at depths of 20, 40 and 60 mm from each horse before (month 0), during (month 3) and after (month 8) training, and again after 3 months of detraining (month 11). MHC composition was analysed by electrophoresis and immunohistochemistry with anti-MHC monoclonal antibodies. Fibre areas, oxidative capacity and capillaries were studied histochemically. The activities of key muscle enzymes of aerobic (citrate synthase and 3-hydroxy-acyl-CoA-dehydrogenase) and anaerobic (phosphofructokinase and lactic dehydrogenase) metabolism and the intramuscular glycogen and triglyceride contents were also biochemically analysed. Early changes with training (3 months) included hypertrophy of type IIA fibres, a reduction of MHC-IIX with a concomitant increase of MHC-IIA, a rise in the number of high-oxidative fibres and in the activities of aerobic muscle enzymes and glycogen content. Long-term changes with training (8 months) were a further decline in the expression of MHC-IIX, an increase of slow MHC-I, additional increases of high-oxidative fibres, capillary density, activities of aerobic enzymes and endogenous glycogen; intramuscular lipid deposits also increased after 8 months of training whereas the activities of anaerobic enzymes declined. Most of exercise-induced alterations reverted after 3 months of detraining. These results indicate that endurance-exercise training induces a reversible transition of MHC composition in equine muscle in the order IIX-->IIA-->I, which is coordinated with changes in the metabolic properties of the muscle. Furthermore, a dose-response relationship was evident between the duration (in total) of training and the magnitude of muscle adaptations.