Differential effects of arginine, glutamate and phosphoarginine on Ca2+-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor- coactivator-1 (ET 8.5-fold, ST 10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET 26-fold, ST 39-fold), vascular endothelial growth factor (VEGF; ET 4.5-fold, ST 4-fold), and muscle atrophy F-box protein (MAFbx) (ET 2-fold, ST 0.4-fold) mRNA increased in both groups, whereas MyoD (3-fold), myogenin (0.9-fold), and myostatin (2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (7-fold, P < 0.01) and MyoD (0.7-fold) increased, whereas MAFbx (0.7-fold) and myostatin (0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Mitofusions1/2 and ERRα expression are increased in human skeletal muscle after physical exercise.

Mitochondrial impairment is hypothesized to contribute to the pathogenesis of insulin resistance. Mitofusin (Mfn) proteins regulate the biogenesis and maintenance of the mitochondrial network, and when inactivated, cause a failure in the mitochondrial architecture and decreases in oxidative capacity and glucose oxidation. Exercise increases muscle mitochondrial content, size, oxidative capacity and aerobic glucose oxidation. To address if Mfn proteins are implicated in these exercise-induced responses, we measured Mfn1 and Mfn2 mRNA levels, pre-, post-, 2 and 24 h post-exercise. Additionally, we measured the expression levels of transcriptional regulators that control mitochondrial biogenesis and functions, including PGC-1α, NRF-1, NRF-2 and the recently implicated ERRα. We show that Mfn1, Mfn2, NRF-2 and COX IV mRNA were increased 24 h post-exercise, while PGC-1α and ERRα mRNA increased 2 h post-exercise. Finally, using in vitro cellular assays, we demonstrate that Mfn2 gene expression is driven by a PGC-1α programme dependent on ERRα. The PGC-1α/ERRα-mediated induction of Mfn2 suggests a role of these two factors in mitochondrial fusion. Our results provide evidence that PGC-1α not only mediates the increased expression of oxidative phosphorylation genes but also mediates alterations in mitochondrial architecture in response to aerobic exercise in humans.

Identification of secreted proteins associated with obesity and type 2 diabetes in Psammomys obesus

Objective: Skeletal muscle produces a variety of secreted proteins that have important roles in intercellular communication and affects processes such as glucose homoeostasis. The objective of this study was to develop a novel Signal Sequence Trap (SST) in conjunction with cDNA microarray technology to identify proteins secreted from skeletal muscle of Psammomys obesus that were associated with obesity and type 2 diabetes (T2D).

Design: Secreted proteins that were differentially expressed between lean, normal glucose tolerant (NGT), overweight and impaired glucose tolerant (IGT) and obese, T2D P. obesus were isolated using SST in conjunction with cDNA microarray technology. Subsequent gene expression was measured in tissues from P. obesus by real-time PCR (RT-PCR).

Results: The SST yielded 1600 positive clones, which were screened for differential expression. A total of 91 (B6%) clones were identified by microarray to be differentially expressed between NGT, IGT and T2D P. obesus. These clones were sequenced to identify 51 genes, of which only 27 were previously known to encode secreted proteins. Three candidate genes not previously associated with obesity or type 2 diabetes, sushi domain containing 2, collagen and calcium-binding EGF domains 1 and periostin (Postn), as well as one gene known to be associated, complement component 1, were shown by RT-PCR to be differentially expressed in  skeletal muscle of P. obesus. Further characterization of the secreted protein Postn revealed it to be predominantly expressed in adipose tissue, with higher expression in visceral compared with subcutaneous adipose depots.

Conclusion: SST in conjunction with cDNA microarray technology is a powerful tool to identify differentially expressed secreted proteins involved in complex diseases such as obesity and type 2 diabetes. Furthermore, a number of candidate genes were identified, in particular, Postn, which may have a role in the development of obesity and type 2 diabetes.

ß2-Agonist administration increases sarcoplasmic reticulum Ca2+-ATPase activity in aged rat skeletal muscle

Aging is associated with a slowing of skeletal muscle contractile properties, including a decreased rate of relaxation. In rats, the age-related decrease in the maximal rate of relaxation is reversed after 4-wk administration with the β2-adrenoceptor agonist (β2-agonist) fenoterol. Given the critical role of the sarcoplasmic reticulum (SR) in regulating intracellular Ca2+ transients and ultimately the time course of muscle contraction and relaxation, we tested the hypothesis that the mechanisms of action of fenoterol are mediated by alterations in SR proteins. Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) kinetic properties were assessed in muscle homogenates and enriched SR membranes isolated from the red (RG) and white (WG) portions of the gastrocnemius muscle in adult (16 mo) and aged (28 mo) F344 rats that had been administered fenoterol for 4 wk (1.4 mg/kg/day ip, in saline) or vehicle only. Aging was associated with a 29% decrease in the maximal activity (Vmax) of SERCA in the RG but not in the WG muscles. Fenoterol treatment increased the Vmax of SERCA and SERCA1 protein levels in RG and WG. In the RG, fenoterol administration reversed an age-related selective nitration of the SERCA2a isoform. Our findings demonstrate that the mechanisms underlying age-related changes in contractile properties are fiber type dependent, whereas the effects of fenoterol administration are independent of age and fiber type.

Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca²⁺- and Sr²⁺-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had ∼10% of the maximal force producing capacity (P_o) of control (uninjured) fibres, and an altered sensitivity to Ca²⁺ and Sr²⁺ at 7 days post-injury. Increased force production and a shift in Ca²⁺ sensitivity consistent with fibre maturation were observed during regeneration such that P_o was restored to 36–45% of that in control fibres by 21 days, and sensitivity to Ca²⁺ and Sr²⁺ was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.

Gender differences in muscle inflammation after eccentric exercise

Unaccustomed exercise is followed by delayed-onset muscle soreness and morphological changes in skeletal muscle. Animal studies have demonstrated that women have an attenuated response to muscle damage. We studied the effect of eccentric exercise in untrained male (n = 8) and female (n = 8) subjects using a unilateral exercise design [exercise (Ex) and control (Con) legs]. Plasma granulocyte counts [before (Pre) and 48 h after exercise (+48h)] and creatine kinase activity [Pre, 24 h after exercise (+24h), +48h, and 6 days after exercise (+6d)] were determined before (Pre) and after (+24h, +48h, +6d) exercise, with biopsies taken from the vastus lateralis of each leg at +48h for determination of muscle damage and/or inflammation. Plasma granulocyte counts increased for men and decreased for women at +48h (P < 0.05), and creatine kinase activity increased for both genders at +48h and +6d (P < 0.01). There were significantly greater areas of both focal (P < 0.001) and extensive (P < 0.01) damage in the Ex vs. Con leg for both genders, which was assessed by using toluidine blue staining. The number of leukocyte common antigen-positive cells/mm2 tissue increased with exercise (P < 0.05), and men tended to show more in their Ex vs. Con leg compared with women (P = 0.052). Men had a greater total (Ex and Con legs) number of bcl-2-positive cells/mm2 tissue vs. women (P < 0.05). Atrophic fibers with homogeneous bcl-2-positive staining were seen only in men (n = 3). We conclude that muscle damage is similar between genders, yet the inflammatory response is attenuated in women vs. men. Finally, exercise may stimulate the expression of proteins involved in apoptosis in skeletal muscle.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Skeletal muscle GLUT-4 gene and protein regulation

Twenty two, young, healthy individuals participated in three studies aiming to assess the effect of various types of physical activity - acute exercise of moderate intensity and duration, varying intensity, short-term training - on skeletal muscle GLUT-4 gene and protein expression as well as on a range of genes encoding the proteins involved in carbohydrate metabolism in skeletal muscle.

Effect of exercise and protein on skeletal muscle inflammatory mediators

Intense exercise results in muscular inflammation. Molecular techniques were used to identify novel inflammatory proteins in human muscle. Males and females displayed different levels of exercise-induced inflammatory proteins. Interestingly, dairy protein supplements reduced these inflammatory proteins post-exercise. Increased dietary red meat consumption, with training, had no impact on muscle inflammation, although strength gain was improved.

Regulation of fat oxidative genes in human skeletal muscle

The ability of skeletal muscle to adapt fat oxidation rates is important for human health. Lipid metabolism requires the involvement of many proteins encoded by their corresponding genes. This thesis demonstrates that manipulating plasma free fatty acid levels alters the expression of selected genes involved in regulating fatty acid metabolism.

Glycogen-protein associations in human skeletal muscle

The collective findings of this dissertation demonstrated little effect of exercise on the absolute or relative expression of glycogen regulatory proteins associated with a glycogen enriched fraction in human skeletal muscle. However the findings of this thesis help inform methodological approaches to future investigations into glycogen-protein associations.

The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy

Skeletal muscle atrophy occurs in many chronic diseases and disuse conditions. Its severity reduces patient recovery, independence and quality of life. The discovery of two muscle-specific E3 ubiquitin ligases, MAFbx/ atrogin-1 and Muscle RING Finger-1 (MuRF1), promoted an expectation of these molecules as targets for therapeutic development. While numerous studies have determined the conditions in which MAFbx/atrogin-1 and MuRF1 mRNA levels are regulated, few studies have investigated their functional role in skeletal muscle. Recently, studies identifying new target substrates for MAFbx/atrogin-1 and
MuRF1, outside of their response to the initiation of muscle atrophy, suggest that there is more to these proteins than
previously appreciated. This review will highlight our present knowledge of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, the impact of potential therapeutics and their known regulators and substrates. Finally, we will comment on new approaches that may expand our knowledge of these two molecules in their control of skeletal muscle function.

TGF-Ý signalling in human skeletal muscle regeneration and diabetes

The TGF-Ý superfamily comprises a large group of proteins with many effects on muscle growth and maturation. The molecular regulation of skeletal muscle regeneration and metabolism in response to prominent superfamily members, myostatin and TGF-Ý1, were analysed, demonstrating the importance of this pathway in controlling how muscles grow and are regulated.

Striated muscle activator of Rho signalling (STARS) is a PGC-1α/oestrogen-related receptor-α target gene and is upregulated in human skeletal muscle after endurance exercise

Exercise improves the ability of skeletal muscle to metabolise fats and sugars. For these improvements to occur the muscle detects a signal caused by exercise, resulting in changes in genes and proteins that control metabolism. We show that endurance exercise increases the amount of a protein called striated muscle activator of Rho signalling (STARS) as well as several other proteins influenced by STARS.We also show that the amount of STARS can be increased by signals directed from proteins called peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) and oestrogen-related receptor-α (ERRα). We also observed that when we reduce the amount of STARS in muscle cells, we block the ability of PGC-1α/ERRα to increase a gene called carnitine palmitoyltransferase-1β (CPT-1β), which is important for fat metabolism. Our study has shown that the STARS pathway is regulated by endurance exercise. STARS may also play a role in fat metabolism in muscle.