Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells.

The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca2+ homeostasis, antioxidant cytoprotection and protein-protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca2+ storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca2+ homeostasis.

Expression of SUMO enzymes is fiber type dependent in skeletal muscles and is dysregulated in muscle disuse.

SUMOylation is a dynamic, reversible, enzymatic drug-targetable post-translational modification (PTM) reaction where the Small Ubiquitin-like Modifier (SUMO) moieties are attached to proteins. This reaction regulates various biological functions like cell growth, differentiation, and it is crucial for maintaining organ homeostasis. However, the actions of SUMO in skeletal muscle pathophysiology are still not investigated. In this study, we quantified the abundance of the SUMO enzymes and determined the distribution of SUMOylated proteins along the fibers of nine different muscles. We find that skeletal muscles contain a distinctive group of SUMO enzymes and SUMOylated proteins in relation to their different metabolism, functions, and fiber type composition. In addition, before the activation of protein degradation pathways, this unique set is quickly altered in response to muscle sedentariness. Finally, we demonstrated that PAX6 acts as an upstream regulator of the SUMO conjugation reaction, which can become a potential therapeutic marker to prevent muscle diseases generated by inactivity.

Reciprocal Homer1a and Homer2 Isoform Expression Is a Key Mechanism for Muscle Soleus Atrophy in Spaceflown Mice.

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.

Chronic Systemic Curcumin Administration Antagonizes Murine Sarcopenia and Presarcopenia.

Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20-35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.

Sarcolemmal loss of active nNOS (Nos1) is an oxidative stress-dependent, early event driving disuse atrophy.

Skeletal muscle atrophy following unloading or immobilization represents a major invalidating event in bedridden patients. Among mechanisms involved in atrophy development, a controversial role is played by neuronal NOS (nNOS; NOS1), whose dysregulation at the protein level and/or subcellular distribution also characterizes other neuromuscular disorders. This study aimed to investigate unloading-induced changes in nNOS before any evidence of myofiber atrophy, using vastus lateralis biopsies obtained from young healthy subjects after a short bed-rest and rat soleus muscles after exposure to short unloading periods. Our results showed that (1) changes in nNOS subcellular distribution using NADPH-diaphorase histochemistry to detect enzyme activity were observed earlier than using immunofluorescence to visualize the protein; (2) loss of active nNOS from the physiological subsarcolemmal localization occurred before myofiber atrophy, i.e. in 8-day bed-rest biopsies and in 6 h-unloaded rat soleus, and was accompanied by increased nNOS activity in the sarcoplasm; (3) nNOS (Nos1) transcript and protein levels decreased significantly in the rat soleus after 6 h and 1 day unloading, respectively, to return to ambulatory levels after 4 and 7 days of unloading, respectively; (4) unloading-induced nNOS redistribution appeared dependent on mitochondrial-derived oxidant species, indirectly measured by tropomyosin disulfide bonds which had increased significantly in the rat soleus already after a 6 h-unloading bout; (5) activity of displaced nNOS molecules is required for translocation of the FoxO3 transcription factor to myofiber nuclei. FoxO3 nuclear localization in rat soleus increased after 6 h unloading (about four-fold the ambulatory level), whereas it did not when nNOS expression and activity were inhibited in vivo before and during 6 h unloading. In conclusion, this study demonstrates that the redistribution of active nNOS molecules from sarcolemma to sarcoplasm not only is ahead of the atrophy of unloaded myofibers, and is induced by increased production of mitochondrial superoxide anion, but also drives FoxO3 activation to initiate muscle atrophy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Ablation of S1P3 receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.

Curcumin counteracts loss of force and atrophy of hindlimb unloaded rat soleus by hampering neuronal nitric oxide synthase untethering from sarcolemma.

Antioxidant administration aimed to antagonize the development and progression of disuse muscle atrophy provided controversial results. Here we investigated the effects of curcumin, a vegetal polyphenol with pleiotropic biological activity, because of its ability to upregulate glucose-regulated protein 94 kDa (Grp94) expression in myogenic cells. Grp94 is a sarco-endoplasmic reticulum chaperone, the levels of which decrease significantly in unloaded muscle. Rats were injected intraperitoneally with curcumin and soleus muscle was analysed after 7 days of hindlimb unloading or standard caging. Curcumin administration increased Grp94 protein levels about twofold in muscles of ambulatory rats (P < 0.05) and antagonized its decrease in unloaded ones. Treatment countered loss of soleus mass and myofibre cross-sectional area by approximately 30% (P ≤ 0.02) and maintained a force-frequency relationship closer to ambulatory levels. Indexes of muscle protein and lipid oxidation, such as protein carbonylation, revealed by Oxyblot, and malondialdehyde, measured with HPLC, were significantly blunted in unloaded treated rats compared to untreated ones (P = 0.01). Mechanistic involvement of Grp94 was suggested by the disruption of curcumin-induced attenuation of myofibre atrophy after transfection with antisense grp94 cDNA and by the drug-positive effect on the maintenance of the subsarcolemmal localization of active neuronal nitric oxide synthase molecules, which were displaced to the sarcoplasm by unloading. The absence of additive effects after combined administration of a neuronal nitric oxide synthase inhibitor further supported curcumin interference with this pro-atrophic pathway. In conclusion, curcumin represents an effective and safe tool to upregulate Grp94 muscle levels and to maintain muscle function during unweighting.

Homer 2 antagonizes protein degradation in slow-twitch skeletal muscles.

Homer represents a new and diversified family of proteins made up of several isoforms. The presence of Homer isoforms, referable to 1b/c and 2a/b, was investigated in fast- and slow-twitch skeletal muscles from both rat and mouse. Homer 1b/c was identical irrespective of the muscle, and Homer 2a/b was instead characteristic of the slow-twitch phenotype. Transition in Homer isoform composition was studied in two established experimental models of atrophy, i.e., denervation and disuse of slow-twitch skeletal muscles of the rat. No change of Homer 1b/c was observed up to 14 days after denervation, whereas Homer 2a/b was found to be significantly decreased at 7 and 14 days after denervation by 70 and 90%, respectively, and in parallel to reduction of muscle mass; 3 days after denervation, relative mRNA was reduced by 90% and remained low thereafter. Seven-day hindlimb suspension decreased Homer 2a/b protein by 70%. Reconstitution of Homer 2 complement by in vivo transfection of denervated soleus allowed partial rescue of the atrophic phenotype, as far as muscle mass, muscle fiber size, and ubiquitinazion are concerned. The counteracting effects of exogenous Homer 2 were mediated by downregulation of MuRF1, Atrogin, and Myogenin, i.e., all genes known to be upregulated at the onset of atrophy. On the other hand, slow-to-fast transition of denervated soleus, another landmark of denervation atrophy, was not rescued by Homer 2 replacement. The present data show that 1) downregulation of Homer 2 is an early event of atrophy, and 2) Homer 2 participates in the control of ubiquitinization and ensuing proteolysis via transcriptional downregulation of MuRF1, Atrogin, and Myogenin. Homers are key players of skeletal muscle plasticity, and Homer 2 is required for trophic homeostasis of slow-twitch skeletal muscles.

Sparing of muscle mass and function by passive loading in an experimental intensive care unit model.

The response to mechanical stimuli, i.e., tensegrity, plays an important role in regulating cell physiological and pathophysiological function, and the mechanical silencing observed in intensive care unit (ICU) patients leads to a severe and specific muscle wasting condition. This study aims to unravel the underlying mechanisms and the effects of passive mechanical loading on skeletal muscle mass and function at the gene, protein and cellular levels. A unique experimental rat ICU model has been used allowing long-term (weeks) time-resolved analyses of the effects of standardized unilateral passive mechanical loading on skeletal muscle size and function and underlying mechanisms. Results show that passive mechanical loading alleviated the muscle wasting and the loss of force-generation associated with the ICU intervention, resulting in a doubling of the functional capacity of the loaded versus the unloaded muscles after a 2-week ICU intervention. We demonstrate that the improved maintenance of muscle mass and function is probably a consequence of a reduced oxidative stress revealed by lower levels of carbonylated proteins, and a reduced loss of the molecular motor protein myosin. A complex temporal gene expression pattern, delineated by microarray analysis, was observed with loading-induced changes in transcript levels of sarcomeric proteins, muscle developmental processes, stress response, extracellular matrix/cell adhesion proteins and metabolism. Thus, the results from this study show that passive mechanical loading alleviates the severe negative consequences on muscle size and function associated with the mechanical silencing in ICU patients, strongly supporting early and intense physical therapy in immobilized ICU patients.

Curcumin Administration Improves Force of mdx Dystrophic Diaphragm by Acting on Fiber-Type Composition, Myosin Nitrotyrosination and SERCA1 Protein Levels.

The vegetal polyphenol curcumin displays beneficial effects against skeletal muscle derangement induced by oxidative stress, disuse or aging. Since oxidative stress and inflammation are involved in the progression of muscle dystrophy, the effects of curcumin administration were investigated in the diaphragm of mdx mice injected intraperitoneally or subcutaneously with curcumin for 4-12-24 weeks. Curcumin treatment independently of the way and duration of administration (i) ameliorated myofiber maturation index without affecting myofiber necrosis, inflammation and degree of fibrosis; (ii) counteracted the decrease in type 2X and 2B fiber percentage; (iii) increased about 30% both twitch and tetanic tensions of diaphragm strips; (iv) reduced myosin nitrotyrosination and tropomyosin oxidation; (v) acted on two opposite nNOS regulators by decreasing active AMP-Kinase and increasing SERCA1 protein levels, the latter effect being detectable also in myotube cultures from mdx satellite cells. Interestingly, increased contractility, decreased myosin nitrotyrosination and SERCA1 upregulation were also detectable in the mdx diaphragm after a 4-week administration of the NOS inhibitor 7-Nitroindazole, and were not improved further by a combined treatment. In conclusion, curcumin has beneficial effects on the dystrophic muscle, mechanistically acting for the containment of a deregulated nNOS activity.

Master Regulators of Muscle Atrophy: Role of Costamere Components.

The loss of muscle mass and force characterizes muscle atrophy in several different conditions, which share the expression of atrogenes and the activation of their transcriptional regulators. However, attempts to antagonize muscle atrophy development in different experimental contexts by targeting contributors to the atrogene pathway showed partial effects in most cases. Other master regulators might independently contribute to muscle atrophy, as suggested by our recent evidence about the co-requirement of the muscle-specific chaperone protein melusin to inhibit unloading muscle atrophy development. Furthermore, melusin and other muscle mass regulators, such as nNOS, belong to costameres, the macromolecular complexes that connect sarcolemma to myofibrils and to the extracellular matrix, in correspondence with specific sarcomeric sites. Costameres sense a mechanical load and transduce it both as lateral force and biochemical signals. Recent evidence further broadens this classic view, by revealing the crucial participation of costameres in a sarcolemmal "signaling hub" integrating mechanical and humoral stimuli, where mechanical signals are coupled with insulin and/or insulin-like growth factor stimulation to regulate muscle mass. Therefore, this review aims to enucleate available evidence concerning the early involvement of costamere components and additional putative master regulators in the development of major types of muscle atrophy.

Grp94 acts as a mediator of curcumin-induced antioxidant defence in myogenic cells.

Curcumin is a non-toxic polyphenol with pleiotropic activities and limited bioavailability. We investigated whether a brief exposure to low doses of curcumin would induce in the myogenic C2C12 cell line an endoplasmic reticulum (ER) stress response and protect against oxidative stress. A 3-hr curcumin administration (5-10 microM) increased protein levels of the ER chaperone Grp94, without affecting those of Grp78, calreticulin and haeme-oxygenase-1 (HO-1). Exposure of cells to hydrogen peroxide 24 hrs after the curcumin treatment decreased caspase-12 activation, total protein oxidation and translocation of NF-kappaB to the nucleus, compared with untreated cells. Grp94 overexpression, achieved by means of either stable or transient trasfection, induced comparable cytoprotective effects to hydrogen peroxide. The delayed cytoprotection induced by curcumin acted through Grp94, because the curcumin-induced increase in Grp94 expression was hampered by either stable or transient transfection with antisense cDNA; in these latter cells, the extent of total protein oxidation, as well as the translocation of NF-kappaB to the nucleus, and the percentage of apoptotic cells were comparable to those observed in both curcumin-untreated wild-type and empty vector transfected cells. Defining the mechanism(s) by which Grp94 exerts its antioxidant defence, the determination of cytosolic calcium levels in C2C12 cells by fura-2 showed a significantly reduced amount of releasable calcium from intracellular stores, both in conditions of Grp94 overexpression and after curcumin pre-treatment. Therefore, a brief exposure to curcumin induces a delayed cytoprotection against oxidative stress in myogenic cells by increasing Grp94 protein level, which acts as a regulator of calcium homeostasis.

Effects of inactivity on human muscle glutathione synthesis by a double-tracer and single-biopsy approach.

Oxidative stress is often associated to inactivity-mediated skeletal muscle atrophy. Glutathione is one of the major antioxidant systems stimulated, both at muscular and systemic level, by activation of oxidative processes. We measured changes in glutathione availability, oxidative stress induction and the extent of atrophy mediated by 35 days of experimental bed rest in vastus lateralis muscle of healthy human volunteers. To assess muscle glutathione synthesis, we applied a novel single-biopsy and double-tracer ([(2)H(2)]glycine and [(15)N]glycine) approach based on evaluation of steady-state precursor incorporation in product. The correlations between the traditional (multiple-samples, one-tracer) and new (one-sample, double-tracer infusion) methods were analysed in erythrocytes by Passing-Bablok and Altman-Bland tests. Muscle glutathione absolute synthesis rate increased following bed rest from 5.5 ± 1.1 to 11.0 ± 1.5 mmol (kg wet tissue)(-1) day(-1) (mean ± S.E.M.; n = 9; P = 0.02) while glutathione concentration failed to change significantly. Bed rest induced vastus lateralis muscle atrophy, as assessed by pennation angle changes measured by ultrasonography (from 18.6 ± 1.0 to 15.3 ± 0.9 deg; P = 0.01) and thickness changes (from 2.3 ± 0.2 to 1.9 ± 0.1 cm; P < 0.001). Moreover, bed rest increased protein oxidative stress, as measured by muscle protein carbonylation changes (from 0.6 ± 0.1 to 1.00 ± 0.1 Oxydized-to-total protein ratio; P < 0.04). In conclusion, we developed in erythrocytes a new minimally invasive method to determine peptide synthesis rate in human tissues. Application of the new method to skeletal muscle suggests that disuse atrophy is associated to oxidative stress induction as well as to compensatory activation of the glutathione system.

Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts.

The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.

Loss of melusin is a novel, neuronal NO synthase/FoxO3-independent master switch of unloading-induced muscle atrophy.

BACKGROUND: Unloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle-specific integrin-associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes. METHODS: Muscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail-suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno-associated virus infection. In vivo interference of exogenous melusin with dominant-negative kinases and other atrophy attenuators (Grp94 cDNA; 7-nitroindazole) on size of unloaded rat myofibers was also explored. RESULTS: Unloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail-suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of ß1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (-31%), compared with controls (-48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin-expressing adeno-associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (-33%) by reducing Atrogin-1 and MurF-1 transcripts (P ≤ 0.002), despite of a two-fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co-transfection with dominant-negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7-nitroindazole treatment or with cDNA transfection of the nNOS-interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy. CONCLUSIONS: Disuse/unloading-induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.

A transient antioxidant stress response accompanies the onset of disuse atrophy in human skeletal muscle.

It is presently unknown whether oxidative stress increases in disused skeletal muscle in humans. Markers of oxidative stress were investigated in biopsies from the vastus lateralis muscle, collected from healthy subjects before [time 0 (T0)], after 1 wk (T8), and after 5 wk (T35) of bed rest. An 18% decrease in fiber cross-sectional area was detected in T35 biopsies (P<0.05). Carbonylation of muscle proteins significantly increased about twofold at T35 (P<0.02) and correlated positively with the decrease in fiber cross-sectional area (P=0.04). Conversely, T8 biopsies showed a significant increase in protein levels of heme oxygenase-1 and glucose-regulated protein-75 (Grp75)/mitochondrial heat shock protein-70, two stress proteins involved in the antioxidant defense (P<0.05). Heme oxygenase-1 increase, which involved a larger proportion of slow fibers compared with T0, appeared blunted in T35 biopsies. Grp75 protein level increased threefold in T8 biopsies and localized especially in slow fibers (P<0.025), to decrease significantly in T35 biopsies (P<0.05). Percent change in Grp75 levels positively correlated with fiber cross-sectional area (P=0.01). Parallel investigations on rat soleus muscles, performed after 1-15 days of hindlimb suspension, showed that Grp75 protein levels significantly increased after 24 h of unloading (P = 0.02), i.e., before statistically significant evidence of muscle atrophy, to decrease thereafter in relation to the degree of muscle atrophy (P=0.03). Therefore, in humans as in rodents, disuse muscle atrophy is characterized by increased protein carbonylation and by the blunting of the antioxidant stress response evoked by disuse.

Cellular distribution of Hsp70 expression in rat skeletal muscles. Effects of moderate exercise training and chronic hypoxia.

Rat hindlimb muscles constitutively express the inducible heat shock protein 72 (Hsp70), apparently in proportion to the slow myosin content. Since it remains controversial whether chronic Hsp70 expression reflects the overimposed stress, we investigated Hsp70 cellular distribution in fast muscles of the posterior rat hindlimb after (1) mild exercise training (up to 30 m/min treadmill run for 1 h/day), which induces a remodeling in fast fiber composition, or (2) prolonged exposure to normobaric hypoxia (10%O(2)), which does not affect fiber-type composition. Both conditions increased significantly protein Hsp70 levels in the skeletal muscle. Immunohistochemistry showed the labeling for Hsp70 in subsets of both slow/type 1 and fast/type 2A myofibers of control, sedentary, and normoxic rats. Endurance training increased about threefold the percentage of Hsp70-positive myofibers (P < 0.001), and changed the distribution of Hsp70 immunoreactivity, which involved a larger subset of both type 2A and intermediate type 2A/2X myofibers (P < 0.001) and vascular smooth muscle cells. Hypoxia induced Hsp70 immunoreactivity in smooth muscle cells of veins and did not increase the percentage of Hsp70-positive myofibers; however, sustained exposure to hypoxia affected the distribution of Hsp70 immunoreactivity, which appeared detectable in a very small subset of type 2A fibers, whereas it concentrated in type 1 myofibers (P < 0.05) together with the labeling for heme-oxygenase isoform 1, a marker of oxidative stress. Therefore, the chronic induction of Hsp70 expression in rat skeletal muscles is not obligatory related to the slow fiber phenotype but reveals the occurrence of a stress response.

Cell stress response in skeletal muscle myofibers.

Cells respond to conditions that impair homeostasis through ex novo synthesis of stress proteins, which differ in subcellular localization and biological function and whose differential expression depends on the type of the stressing stimulus and on the involvement of the specific stress-response signaling cascade. The biological significance of such an event is the increased resistance against further perturbations of cell homeostasis, and thus, enhanced survival. We will review briefly the available evidence concerning stress response of skeletal muscle cells, including recent results indicating the involvement of endoplasmic reticulum stress response and proteins in skeletal muscle cell differentiation and in progression of muscle diseases.

Troponin I isoform expression in human and experimental atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is accompanied by re-expression of fetal genes and activation of proteolytic enzymes. In this study both aspects were addressed with respect to troponin I (TnI) isoform expression. METHODS AND RESULTS: Western blotting and real-time reverse transcription-polymerase chain reaction were used to study TnI isoform expression in patients with paroxysmal or chronic AF and in goats after 1, 2, 4, 8, and 16 weeks of AF. In addition to cardiac TnI (cTnI), low expression of slow-twitch skeletal TnI (ssTnI) protein was found in 60% of patients in sinus rhythm or paroxysmal AF and in 8% of patients with chronic AF. In adult goat atrium, ssTnI protein expression was undetectable. Calcium-dependent degradation of cTnI protein was found in 1 or 2 of 6 animals after 1 to 4 weeks of AF. Although always low, ssTnI mRNA levels were significantly higher in patients who expressed ssTnI protein than in those who did not. Relative ssTnI mRNA expression was significantly lower in patients with paroxysmal AF and chronic AF than in those in sinus rhythm. In goats there was a tendency toward higher relative levels of ssTnI at the onset of AF followed by a normalization when AF had become sustained. CONCLUSIONS: Atrial re-expression of ssTnI during paroxysmal AF in patients and during the first 2 weeks of pacing-induced AF in goats does not seem to be part of the process of AF-associated cardiomyocyte dedifferentiation but seems to result from transient cardiomyocyte stress at the onset of AF.

Overexpression of the stress protein Grp94 reduces cardiomyocyte necrosis due to calcium overload and simulated ischemia.

Increase in free intracellular calcium [Ca 2+]i plays a crucial role in cardiomyocyte ischemic injury. Here we demonstrate that overexpression of the sarcoplasmic-reticulum stress-protein Grp94 reduces myocyte necrosis due to calcium overload or simulated ischemia. Selective three- to eightfold Grp94 increase, with no change in Grp78 or calreticulin amount, was achieved by stable transfection of skeletal C2C12 and cardiac H9c2 muscle cells. After exposure to the calcium ionophore A23187, LDH release from five different Grp94-overexpressing clones of either C2C12 and H9c2 origin was significantly lower than that of control ones and [Ca 2+]i increase was significantly delayed. The number of necrotic cells, evaluated by propidium iodide uptake, was reduced when cells from the Grp94-overexpressing H9c2 clone were exposed to conditions simulating ischemia. Experiments performed in neonatal rat cardiomyocytes co-transfected with grp94 and the green fluorescent protein (GFP) cDNAs validated the protective effect of Grp94 overexpression. A lower percentage of propidium-iodide positive/GFP-fluorescent myocytes co-expressing exogenous Grp94, with respect to myocytes expressing GFP alone, was observed after exposure to either A23187 (6.6% vs. 14.0%, respectively) or simulated ischemia (8.5% vs. 17.7%, respectively). In conclusion, the selective increase in Grp94 protects cardiomyocytes from both ischemia and calcium overload counteracting [Ca 2+]i elevations.