Calpain-3 Is Not a Sodium Dependent Protease and Simply Requires Calcium for Activation.

Calpain-3 (CAPN3) is a muscle-specific member of the calpain family of Ca2+-dependent proteases. It has been reported that CAPN3 can also be autolytically activated by Na+ ions in the absence of Ca2+, although this was only shown under non-physiological ionic conditions. Here we confirm that CAPN3 does undergo autolysis in the presence of high [Na+], but this only occurred if all K+ normally present in a muscle cell was absent, and it did not occur even in 36 mM Na+, higher than what would ever be reached in exercising muscle if normal [K+] was present. CAPN3 in human muscle homogenates was autolytically activated by Ca2+, with ~50% CAPN3 autolysing in 60 min in the presence of 2 µM Ca2+. In comparison, autolytic activation of CAPN1 required about 5-fold higher [Ca2+] in the same conditions and tissue. After it was autolysed, CAPN3 unbound from its tight binding on titin and became diffusible, but only if the autolysis led to complete removal of the IS1 inhibitory peptide within CAPN3, reducing the C-terminal fragment to 55 kDa. Contrary to a previous report, activation of CAPN3, either by raised [Ca2+] or Na+ treatment, did not cause proteolysis of the skeletal muscle Ca2+ release channel-ryanodine receptor, RyR1, in physiological ionic conditions. Treatment of human muscle homogenates with high [Ca2+] caused autolytic activation of CAPN1, accompanied by proteolysis of some titin and complete proteolysis of junctophilin (JP1, full length ~95 kDa), generating an equimolar amount of a diffusible ~75 kDa N-terminal JP1 fragment, but without any proteolysis of RyR1.

Elevated MMP2 abundance and activity in mdx mice are alleviated by prenatal taurine supplementation.

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle-wasting disorder that leads to early death. The mdx mouse is a naturally occurring mutant model for DMD. It lacks dystrophin and displays peak muscle cell necrosis at ~28 days (D28), but in contrast to DMD, mdx mice experience muscle regeneration by D70. We hypothesized that matrix metalloproteinase-2 (MMP2) and/or MMP9 play key roles in the degeneration/regeneration phases in mdx mice. MMP2 abundance in muscle homogenates, measured by calibrated Western blotting, and activity, measured by zymogram, were lower at D70 compared with D28 in both mdx and wild-type (WT) mice. Importantly, MMP2 abundance was higher in both D28 and D70 mdx mice than in age-matched WT mice. The higher MMP2 abundance was not due to infiltrating macrophages, because MMP2 content was still higher in isolated muscle fibers where most macrophages had been removed. Prenatal supplementation with the amino acid taurine, which improved muscle strength in D28 mdx mice, produced approximately twofold lower MMP2 activity, indicating that increased MMP2 abundance is not required when muscle damage is attenuated. There was no difference in MMP9 abundance between age-matched WT and mdx mice (P > 0.05). WT mice displayed decreased MMP9 abundance as they aged. While MMP9 may have a role during age-related skeletal muscle growth, it does not appear essential for degeneration/regeneration cycles in the mdx mouse. Our findings indicate that MMP2 plays a more active role than MMP9 in the degenerative phases of muscle fibers in D28 mdx mice.

Effects of S-glutathionylation on the passive force-length relationship in skeletal muscle fibres of rats and humans.

This study investigated the effect of S-glutathionylation on passive force in skeletal muscle fibres, to determine whether activity-related redox reactions could modulate the passive force properties of muscle. Mechanically-skinned fibres were freshly obtained from human and rat muscle, setting sarcomere length (SL) by laser diffraction. Larger stretches were required to produce passive force in human fibres compared to rat fibres, but there were no fibre-type differences in either species. When fibres were exposed to glutathione disulfide (GSSG; 20 mM, 15 min) whilst stretched (at a SL where passive force reached ~ 20% of maximal Ca2+-activated force, denoted as SL20 % max), passive force was subsequently decreased at all SLs in both type I and type II fibres of rat and human (e.g., passive force at SL20 % max decreased by 12 to 25%). This decrease was fully reversed by subsequent reducing treatment with dithiothreitol (DTT; 10 mM for 10 min). If freshly skinned fibres were initially treated with DTT, there was an increase in passive force in type II fibres (by 10 ± 3% and 9 ± 2% in rat and human fibres, respectively), but not in type I fibres. These results indicate that (i) S-glutathionylation, presumably in titin, causes a decrease in passive force in skeletal muscle fibres, but the reduction is relatively smaller than that reported in cardiac muscle, (ii) in rested muscle in vivo, there appears to be some level of reversible oxidative modification, probably involving S-glutathionylation of titin, in type II fibres, but not in type I fibres.

Distribution and activation of matrix metalloproteinase-2 in skeletal muscle fibers.

A substantial intracellular localization of matrix metalloproteinase 2 (MMP2) has been reported in cardiomyocytes, where it plays a role in the degradation of the contractile apparatus following ischemia-reperfusion injury. Whether MMP2 may have a similar function in skeletal muscle is unknown. This study determined that the absolute amount of MMP2 is similar in rat skeletal and cardiac muscle and human muscle (~10-18 nmol/kg muscle wet wt) but is ~50- to 100-fold less than the amount of calpain-1. We compared mechanically skinned muscle fibers, where the extracellular matrix (ECM) is completely removed, with intact fiber segments and found that ~30% of total MMP2 was associated with the ECM, whereas ~70% was inside the muscle fibers. Concordant with whole muscle fractionation, further separation of skinned fiber segments into cytosolic, membranous, and cytoskeletal and nuclear compartments indicated that ~57% of the intracellular MMP2 was freely diffusible, ~6% was associated with the membrane, and ~37% was bound within the fiber. Under native zymography conditions, only 10% of MMP2 became active upon prolonged (17 h) exposure to 20 µM Ca2+, a concentration that would fully activate calpain-1 in seconds to minutes; full activation of MMP2 would require ~1 mM Ca2+. Given the prevalence of intracellular MMP2 in skeletal muscle, it is necessary to investigate its function using physiological conditions, including isolation of any potential functional relevance of MMP2 from that of the abundant protease calpain-1.

Skeletal muscle fibre swelling contributes to force depression in rats and humans: a mechanically-skinned fibre study.

This study investigated the effects of fibre swelling on force production in rat and human skinned muscle fibres, using osmotic compression to reverse the fibre swelling. In mechanically-skinned fibres, the sarcolemma is removed but normal excitation-contraction coupling remains functional. Force responses in mechanically-skinned fibres were examined with and without osmotic compression by polyvinylpyrrolidone 40 kDa (PVP-40) or Dextran 500 kDa (dextran). Fibre diameter increased to 116 ± 2% (mean ± SEM) when rat skinned type II fibres were immersed in the standard intracellular solution, but remained close to the in situ size when 3% (mass/volume) PVP-40 or 4% Dextran were present. Myofibrillar Ca2+ sensitivity, as indicated by pCa50 (- log10[Ca2+] at half-maximal force), was increased in 4% Dextran (0.072 ± 0.007 pCa50 shift), but was not significantly changed in 3% PVP-40. Maximum Ca2+-activated force increased slightly to 103 ± 1% and 104 ± 1% in PVP-40 and Dextran, respectively. Both tetanic and depolarization-induced force responses in rat skinned type II fibres, elicited by electrical stimulation and ion substitution respectively, were increased by ~ 10 to 15% when the fibres were returned to their normal in situ diameter by addition of PVP-40 or Dextran. Interestingly, the potentiation of these force responses in PVP-40 was appreciably greater than could be explained by potentiation of myofibrillar function alone. These results indicate that muscle fibre swelling, as can occur with intense exercise, decreases evoked force responses by reducing both the Ca2+-sensitivity of the contractile apparatus properties and Ca2+ release from the sarcoplasmic reticulum.

Physiological and biochemical characteristics of skeletal muscles in sedentary and active rats.

Laboratory rats are sedentary if housed in conditions where activity is limited. Changes in muscle characteristics with chronic inactivity were investigated by comparing sedentary rats with rats undertaking voluntary wheel running for either 6 or 12 weeks. EDL (type II fibers) and soleus (SOL) muscles (predominantly type I fibers) were examined. When measured within 1-2 h post-running, calcium sensitivity of the contractile apparatus was increased, but only in type II fibers. This increase disappeared when fibers were treated with DTT, indicative of oxidative regulation of the contractile apparatus, and was absent in fibers from rats that had ceased running 24 h prior to experiments. Specific force production was ~ 10 to 25% lower in muscle fibers of sedentary compared to active rats, and excitability of skinned fibers was decreased. Muscle glycogen content was ~ 30% lower and glycogen synthase content ~ 50% higher in SOL of sedentary rats, and in EDL glycogenin was 30% lower. Na+, K+-ATPase α1 subunit density was ~ 20% lower in both EDL and SOL in sedentary rats, and GAPDH content in SOL ~ 35% higher. There were no changes in content of the calcium handling proteins calsequestrin and SERCA, but the content of CSQ-like protein was increased in active rats (by ~ 20% in EDL and 60% in SOL). These findings show that voluntary exercise elicits an acute oxidation-induced increase in Ca2+ sensitivity in type II fibers, and also that there are substantial changes in skeletal muscle characteristics and biochemical processes in sedentary rats.

Effect of androgen deprivation therapy on the contractile properties of type I and type II skeletal muscle fibres in men with non-metastatic prostate cancer.

The contractile properties of vastus lateralis muscle fibres were examined in prostate cancer (PrCa) patients undergoing androgen deprivation therapy (ADT) and in age- and activity-matched healthy male subjects (Control). Mechanically-skinned muscle fibres were exposed to a sequence of heavily Ca2+ -buffered solutions at progressively higher free [Ca2+ ] to determine their force-Ca2+ relationship. Ca2+ -sensitivity was decreased in both type I and type II muscle fibres of ADT subjects relative to Controls (by -0.05 and -0.04 pCa units, respectively, P < .02), and specific force was around 13% lower in type I fibres of ADT subjects than in Controls (P = .02), whereas there was no significant difference in type II fibres. Treatment with the reducing agent dithiothreitol slightly increased specific force in type I and type II fibres of ADT subjects (by ~2%-3%, P < .05) but not in Controls. Pure type IIx fibres were found frequently in muscle from ADT subjects but not in Controls, and the overall percentage of myosin heavy chain IIx in muscle samples was 2.5 times higher in ADT subjects (P < .01). The findings suggest that testosterone suppression can negatively impact the contractile properties by (i) reducing Ca2+ -sensitivity in both type I and type II fibres and (ii) reducing maximum specific force in type I fibres.

Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Changes in contractile and metabolic parameters of skeletal muscle as rats age from 3 to 12 months.

Laboratory rats are considered mature at 3 months despite that musculoskeletal growth is still occurring. Changes in muscle physiological and biochemical characteristics during development from 3 months, however, are not well understood. Whole muscles and single skinned fibres from fast-twitch extensor digitorum longus (EDL) and predominantly slow-twitch soleus (SOL) muscles were examined from male Sprague-Dawley rats (3, 6, 9, 12 months). Ca2+ sensitivity of contractile apparatus decreased with age in both fast- (~ 0.04 pCa units) and slow-twitch (~ 0.07 pCa units) muscle fibres, and specific force increased (by ~ 50% and ~ 25%, respectively). Myosin heavy chain composition of EDL and SOL muscles altered to a small extent with age (decrease in MHCIIa proportion after 3 months). Glycogen content increased with age (~ 80% in EDL and 25% in SOL) and GLUT4 protein density decreased (~ 35 and 20%, respectively), whereas the glycogen-related enzymes were little changed. GAPDH protein content was relatively constant in both muscle types, but COXIV protein decreased ~ 40% in SOL muscle. Calsequestrin (CSQ) and SERCA densities remained relatively constant with age, whereas there was a progressive ~ 2-3 fold increase in CSQ-like proteins, though their role and importance remain unclear. There was also ~ 40% decrease in the density of the Na+, K+-ATPase (NKA) α1 subunit in EDL and the α2 subunit in SOL. These findings emphasise there are substantial changes in skeletal muscle function and the density of key proteins during early to mid-adulthood in rats, which need to be considered in the design and interpretation of experiments.

When phosphorylated at Thr148, the ß2-subunit of AMP-activated kinase does not associate with glycogen in skeletal muscle.

The 5'-AMP-activated protein kinase (AMPK), a heterotrimeric complex that functions as an intracellular fuel sensor that affects metabolism, is activated in skeletal muscle in response to exercise and utilization of stored energy. The diffusibility properties of α- and ß-AMPK were examined in isolated skeletal muscle fiber segments dissected from rat fast-twitch extensor digitorum longus and oxidative soleus muscles from which the surface membranes were removed by mechanical dissection. After the muscle segments were washed for 1 and 10 min, ∼60% and 75%, respectively, of the total AMPK pools were found in the diffusible fraction. After in vitro stimulation of the muscle, which resulted in an ∼80% decline in maximal force, 20% of the diffusible pool became bound in the fiber. This bound pool was not associated with glycogen, as determined by addition of a wash step containing amylase. Stimulation of extensor digitorum longus muscles resulted in 28% glycogen utilization and a 40% increase in phosphorylation of the downstream AMPK target acetyl carboxylase-CoA. This, however, had no effect on the proportion of total ß2-AMPK that was phosphorylated in whole muscle homogenates measured by immunoprecipitation. These findings suggest that, in rat skeletal muscle, ß2-AMPK is not associated with glycogen and that activation of AMPK by muscle contraction does not dephosphorylate ß2-AMPK. These findings question the physiological relevance of the carbohydrate-binding function of ß2-AMPK in skeletal muscle.

Interplay between Mg2+ and Ca2+ at multiple sites of the ryanodine receptor.

RyR1 is an intracellular Ca2+ channel important in excitable cells such as neurons and muscle fibers. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 Å resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.

Important considerations for protein analyses using antibody based techniques: down-sizing Western blotting up-sizes outcomes.

Western blotting has been used for protein analyses in a wide range of tissue samples for >30 years. Fundamental to Western blotting success are a number of important considerations, which unfortunately are often overlooked or not appreciated. Firstly, lowly expressed proteins may often be better detected by dramatically reducing the amount of sample loaded. Single cell (fibre) Western blotting demonstrates the ability to detect proteins in small sample sizes, 5-10 µg total mass (1-3 µg total protein). That is an order of magnitude less than often used. Using heterogeneous skeletal muscle as the tissue of representation, the need to undertake Western blotting in sample sizes equivalent to single fibre segments is demonstrated. Secondly, incorrect results can be obtained if samples are fractionated and a proportion of the protein of interest inadvertently discarded during sample preparation. Thirdly, quantitative analyses demand that a calibration curve be used. This is regardless of using a loading control, which must be proven to not change with the intervention and also be appropriately calibrated. Fourthly, antibody specificity must be proven using whole tissue analyses, and for immunofluorescence analyses it is vital that only a single protein is detected. If appropriately undertaken, Western blotting is reliable, quantitative, both in relative and absolute terms, and extremely valuable.

Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat.

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ∼1.1 and 4.6 µmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (∼3.4 and ∼8.9 µmol/kg, respectively). αB-crystallin content of EDL muscle was ∼4.9 µmol/kg but in SOL muscle was ∼30-fold higher (∼140 µmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ∼95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers.

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ∼95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.

Acute effects of reactive oxygen and nitrogen species on the contractile function of skeletal muscle.

Reactive oxygen and nitrogen species (ROS/RNS) are important for skeletal muscle function under both physiological and pathological conditions. ROS/RNS induce long-term and acute effects and the latter are the focus of the present review. Upon repeated muscle activation both oxygen and nitrogen free radicals likely increase and acutely affect contractile function. Although fluorescent indicators often detect only modest increases in ROS during repeated activation, there are numerous studies showing that manipulations of ROS can affect muscle fatigue development and recovery. Exposure of intact muscle fibres to the oxidant hydrogen peroxide (H(2)O(2)) affects mainly the myofibrillar function, where an initial increase in Ca(2+) sensitivity is followed by a decrease. Experiments on skinned fibres show that these effects can be attributed to H(2)O(2) interacting with glutathione and myoglobin, respectively. The primary RNS, nitric oxide (NO()), may also acutely affect myofibrillar function and decrease the Ca(2+) sensitivity. H(2)O(2) can oxidize the sarcoplasmic reticulum Ca(2+) release channels. This oxidation has a large stimulatory effect on Ca(2+)-induced Ca(2+) release of isolated channels, whereas it has little or no effect on the physiological, action potential-induced Ca(2+) release in skinned and intact muscle fibres. Thus, acute effects of ROS/RNS on muscle function are likely to be mediated by changes in myofibrillar Ca(2+) sensitivity, which can contribute to the development of muscle fatigue or alternatively help counter it.

Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice.

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 µmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 µmol/kg (with SR Ca2+ likely<250 µmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.

Nuclei isolation methods fail to accurately assess the subcellular localization and behaviour of proteins in skeletal muscle.

AIM: Subcellular fractionation is often used to determine the subcellular localization of proteins, including whether a protein translocates to the nucleus in response to a given stimulus. Examining nuclear proteins in skeletal muscle is difficult because myonuclear proteins are challenging to isolate unless harsh treatments are used. This study aimed to determine the most effective method for isolating and preserving proteins in their native state in skeletal muscle. METHODS: We compared the ability of detergents, commercially available kit-based and K+ -based physiological methodologies for isolating myonuclear proteins from resting samples of human muscle by determining the presence of marker proteins for each fraction by western blot analyses. RESULTS: We found that following the initial pelleting of nuclei, treatment with 1% Triton-X 100, 1% CHAPS or 0.5% Na-deoxycholate under various ionic conditions resulted in the nuclear proteins being either resistant to isolation or the proteins present behaving aberrantly. The nuclear proteins in brain tissue were also resistant to 1% Triton-X 100 isolation. Here, we demonstrate aberrant behaviour and erroneous localization of proteins using the kit-based method. The aberrant behaviour was the activation of Ca2+ -dependent protease calpain-3, and the erroneous localization was the presence of calpain-3 and troponin I in the nuclear fraction. CONCLUSION: Our findings indicate that it may not be possible to reliably determine the translocation of proteins between subcellular locations and the nucleus using subcellular fractionation techniques. This study highlights the importance of validating subcellular fractionation methodologies using several subcellular-specific markers and solutions that are physiologically relevant to the intracellular milieu.

Expression of titin-linked putative mechanosensing proteins in skeletal muscle after power resistance exercise in resistance-trained men.

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex + Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P ≤ 0.05) pHSP27-Ser15 (∼6-fold) and creatine kinase (∼50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (∼15-fold) and MLP (∼2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (∼3-fold, Ex) and MLPb (∼20-fold, Ex + Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.NEW & NOTEWORTHY This is the first study to assess the early changes in the expression of titin-linked putative mechanosensing proteins and associated myogenic regulatory factors in skeletal muscle after power resistance exercise in fed, resistance-trained men. We report that power resistance exercise induces subtle early responses in the expression of Ankrd 1 and MLP, suggesting these proteins play a role in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.