Articles in PresS. J Appl Physiol (April 3, 2014). doi:10.1152/japplphysiol.01026.2013 1

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Small heat shock proteins translocate to the cytoskeleton in human skeletal muscle

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following eccentric exercise independently of phosphorylation

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Noni T. Frankenberg1, Graham D. Lamb1, Kristian Overgaard2, Robyn M. Murphy1*

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and Kristian Vissing2

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Department of Zoology, La Trobe University, Melbourne, Victoria, 3086, Australia; 2

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Section of Sport Science, Dept. of Public Health, Aarhus University, DK-8000 Aarhus,

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Denmark.

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*Corresponding author:

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Robyn M. Murphy Department of Zoology La Trobe University Victoria 3086 Australia Email: [email protected] Phone: +61-3-9479 2302 Fax: +61-3-9479 1551

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Keywords: HSP27; αB-crystallin, phosphorylation, eccentric exercise; single fibers

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Running head: phosphorylated sHSP and eccentric exercise in humans

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Copyright © 2014 by the American Physiological Society.

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Abstract

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Small heat shock proteins (sHSPs) are a subgroup of the highly conserved family of HSPs

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that are stress inducible and confer resistance to cellular stress and injury. This study aimed

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to quantitatively examine whether type of contraction (concentric or eccentric) affects sHSPs,

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HSP27 and αB-crystallin, localization and phosphorylation in human muscle. Vastus lateralis

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muscle biopsies from 11 healthy male volunteers were obtained pre, 3h, 24h and 7d following

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concentric (CONC), eccentric (ECC1) and repeated bout eccentric (ECC2) exercise. No

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changes were apparent in a control group (n=5) who performed no exercise. Eccentric

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exercise induced muscle damage, as evidenced by increased muscle force loss, perceived

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muscle soreness, and elevated plasma creatine kinase and myoglobin levels. Total HSP27

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and αB-crystallin amounts did not change following any type of exercise. Following

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eccentric exercise (ECC1 and ECC2) phosphorylation of HSP27 at serine 15 (pHSP27-Ser15)

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was increased ~3-6 fold at 3h, and pαB-crystallin-Ser59 increased ~10-fold at 3h. Prior to

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exercise most of the sHSP and psHSP pools were present in the cytosolic compartment.

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Eccentric exercise resulted in partial redistribution of HSP27 (~23%) from the cytosol to the

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cytoskeletal fraction (~28% for pHSP27-Ser15 and ~7% for pHSP27-Ser82), with subsequent

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full reversal within 24h. αB-crystallin also showed partial redistribution from the cytosolic to

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cytoskeletal fraction (~18% of total) 3h post ECC1, but not after ECC2. There was no

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redistribution or phosphorylation of sHSPs with CONC. Eccentric exercise results in

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increased sHSP phosphorylation and translocation to the cytoskeletal fraction, but the sHSP

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translocation is not dependent on their phosphorylation.

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Introduction

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Heat shock proteins (HSPs) are a highly conserved family of proteins that are stress inducible

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and confer resistance to various forms of cellular stress and injury (26). Small heat shock

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proteins (sHSP) are a distinct subclass of HSPs characterized by a conserved c-terminal

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crystallin domain. HSP27 (homologous to rodent HSP25) and αB-crystallin, also known as

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HspB1 and HspB5 respectively, are the most widely distributed members of the sHSP family,

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being constitutively expressed in many cell types. In muscle, an important function of

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HSP27 and αB-crystallin seems to be the ability to stabilize the cytoskeleton and myofibrils,

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as they appear to prevent denatured proteins from irreversibly aggregating (3, 11). In

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addition to this sHSPs are seemingly involved in the regulation of the cellular redox state (17)

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and inhibition of cellular apoptotic pathway (12, 30).

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In skeletal muscle, as in other organs, exposure to stressful insults can lead to the

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development of stress tolerance, meaning that it can bestow resistance to a second potentially

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damaging stimulus. Eccentric exercise (i.e. where contracting muscles are lengthened)

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constitutes one such stress. In unaccustomed individuals, eccentric exercise can result in

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membrane disruption, myofibrillar disorganization, increased intracellular Ca2+ and

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prolonged decrease in force generating capacity (10, 21). There are multiple effectors that

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enable muscle cells to adapt to exercise and recurrent stress events, with studies in rodents

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showing the sHSPs likely having an important role (15). It is evident that the response of

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sHSPs is dependent on the type and intensity of the exercise. In humans, eccentric exercise

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has been reported to result in up-regulation in the amounts of the sHSPs (6, 28, 29, 31) and

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also in their translocation (27). In quiescent rat muscle the great majority of HSP25/HSP27

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and αB-crystallin are present as freely diffusible proteins (1, 16, 18). This changes however

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if the muscle is subjected to stresses such as increased temperature (19) or eccentric

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contractions in rat (15, 16), cardiac cells (30) and human (31), with the sHSPs translocating

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to the Z-disk region following eccentric contractions in rat skeletal muscle (16). With

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concentric exercise in humans there appears to be no significant increase in the amounts of

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the sHSP (22, 31), but it remains unclear whether or to what extent there is any sHSP

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translocation.

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A further important aspect is that phosphorylation of HSP27 and αB-crystallin can regulate

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their activities or properties, although these are not well understood. From cell-based studies,

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it has been proposed that phosphorylation of sHSPs increases their cytoprotective effects,

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possibly by reducing the extent of sHSP oligomerization (13, 20), though other studies do not

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support this (14). In rat skeletal muscle, both at rest and following eccentric contractions,

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HSP27 and αB-crystallin was found to exist primarily as monomers rather than in large

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oligomeric complexes (16), suggesting that there is no need for phosphorylation-induced

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de-oligomerization of the sHSPs in skeletal muscle. We have previously shown in rat

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skeletal muscle that heat exposure induces an increase in the level of sHSP phosphorylation

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(at Ser85 on HSP27 and Ser59 on αB-crystallin) and that a large proportion of the

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phosphorylated sHSP become bound within the fibers (19), but there was no evidence that the

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phosphorylated sHSPs became bound any differently from non-phosphorylated sHSPs. Koh

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and Escobedo reported that eccentric contractions in rat skeletal muscle also resulted in

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increased phosphorylation of the sHSPs and movement of most of the sHSPs from the

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soluble/cytosolic fraction to the insoluble/cytoskeletal fraction (16). In regard to human

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skeletal muscle, there are no reports to date either on the phosphorylation state of particular

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sHSP Ser residues or on the localization of phosphorylated HSP27 and αB-crystallin in

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exercised muscle.

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In the present study we investigated the amounts and localization of HSP27 and αB-crystallin

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and their phosphorylated forms (HSP27-Ser15 and Ser82 and αB-crystallin-Ser59) in human

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skeletal muscle at rest and following concentric and eccentric exercise. Specifically, we

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examined whether there were changes in the amounts of the sHSP and the phosphorylated

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sHSPs in the muscle following the different types of exercise and whether phosphorylation

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affected the distribution of the sHSP within the muscle cells. Our hypotheses were that 1)

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there would be up-regulation of phosphorylated sHSP protein following muscle-damaging

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eccentric exercise (ECC1), but not concentric exercise (CONC), 2) that the sHSP up-

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regulation and translocation would be attenuated upon a repeated bout of eccentric exercise

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(ECC2), due to there being less muscle damage and hence less stress, and 3) that

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phosphorylated sHSPs would be found predominantly in the cytoskeletal fraction, both before

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and following eccentric exercise.

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Method and materials

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Exercise subjects and protocol

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Details of the exercise protocol and outcomes have been described elsewhere (25) and

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additional results from these experiments appear in the literature (24, 31, 32). All subjects

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were healthy and physically active males, and written consent was obtained from all

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participants. All experiments were carried out in accordance with the declaration of Helsinki

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and approved by the Danish Ethical Committee of Aarhus (J. no. 20040159). Muscle tissue

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from a subsample of those subjects was analyzed in the present study (11 subjects for whom

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full data sets were available and 5 control subjects; mean ± SD; 24 ±3 yr old, 183 ±9 cm

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height, 79 ±8 kg mass). Subjects performed a single 30 min bout of bench-stepping at a

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predetermined step height of 110% of the lower leg length at 60 steps per minute. This

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protocol enables the muscle involved in knee extension to work concentrically (CONC) on

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the step up and the same muscles in the other leg to contract eccentrically (ECC1) on the step

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down using the same leg. The step exercise protocol was then repeated eight weeks after the

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first exercise bout and muscle samples were again obtained from the eccentrically working

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leg (ECC2). No exercise was performed for the Control subjects (n=5). As described

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elsewhere (25, 32), several methods were used to measure the extent to which different

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exercise modalities induced muscle damage. Perceived muscle soreness was 40% greater in

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the ECC1 leg over the CONC leg two days after exercise. In regard to muscle force loss, an

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11-15% reduction was observed 1-2 days after eccentric exercise (ECC1 and ECC2). In the

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subsample of 11 subjects used here, the loss of muscle force was greater 3h following ECC1

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compared with ECC2 (P80% of the HSP27 and

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αB-crystallin from the cytosolic/diffusible fraction to the cytoskeletal/insoluble fraction.

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Significantly, this almost complete relocation of the αB-crystallin occurred despite only

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~20% of the αB-crystallin being phosphorylated, again demonstrating that phosphorylation

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was not necessary for the sHSP relocation. In cardiac muscle, ischemia leads to both

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phosphorylation of sHSP and translocation of sHSPs from the cytosol to the cytoskeleton (2,

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5, 11, 12). However, it was further found that inducing phosphorylation of αB-crystallin with

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agonists did not cause translocation, and it was concluded that phosphorylation was neither

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necessary nor sufficient for translocation of αB-crystallin in cardiac tissue (5). Lastly, it has

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been reported that phosphorylation of HSP27 in Chinese hamster cell lines was necessary to

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confer its full cytoprotective effects to thermal stimuli, and for reducing oligimeric size, but

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was not involved in cellular localization (20).

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Comparison between different phosphorylation sites on HSP27 and their response to exercise

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The stress protocol of eccentric exercise produced differences in phosphorylation of HSP27

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at Ser15 and at Ser82. pHSP27-Ser15 was increased more than 2.5-fold for an extended

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period (3-24 h) following a first bout of eccentric exercise and the exercise also resulted in its

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translocation, whereas there was no significant effect on pHSP27-Ser82. These results

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suggest that phosphorylation at either site might have functions unique from each other or if

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the functions are similar, that they are triggered by unique events.

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Conclusions

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Our results demonstrate that muscle-damaging eccentric exercise results in phosphorylation

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of HSP27 and αB-crystallin, and that less-damaging concentric exercise alone is insufficient

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to phosphorylate the sHSPs and to move them from a predominantly cytosolic localization to

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bound sites within the cell. Translocation of HSP27 and αB-crystallin to the cytoskeleton, as

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well as phosphorylation at specific Ser sites (pHSP27-Ser-15 and pαB-crystallin-Ser-59),

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occur transiently following eccentric exercise. Using the physiologically-relevant exercise

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intervention of the current study, which caused comparatively mild muscle damage, only a

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small percentage of the total pool (~18 to 23%) of sHSPs was found to translocate and bind

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to cytoskeletal sites. The study provides the first evidence that exercise interventions result

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in phosphorylation of the small HSPs in human skeletal muscle, and that this occurs

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independently of the translocation events. Whilst phosphorylation of sHSP may play a

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central role in the function of sHSP, what these function(s) are and what role it has in the

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protection of muscle, remains unclear.

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Acknowledgements

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The monoclonal antibodies directed against adult human MHC isoforms (A4.84 and A4.74)

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used in the present study were developed by Dr Blau, those directed against SERCA1 were

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developed by Dr D Fambrough. All were obtained from the Development Studies

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Hybridoma Bank, under the auspices of the NICHD and maintained by the University of

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Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA. The study was

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supported by National Health and Medical Research Council of Australia (1051460),

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Australian Research Council and The Danish Ministry of Culture (grant no. 2004-05-029).

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Table 1. sHSP and phosphorylated sHSP protein content in control (non-exercised) subjects. Amounts normalized to myosin and expressed relative to Pre sample. Values are means ± SEM, n=5; no significant differences from Pre level. Protein

Pre

3h

24 h

7d

HSP27 pHSP27-Ser15 pHSP27-Ser82

1.0 ± 0.3 1.0 ± 0.3 1.0 ± 0.2

0.9 ± 0.3 0.6 ± 0.3 1.5 ± 0.5

1.0 ± 0.3 0.9 ± 0.3 1.0 ± 0.2

1.1 ± 0.5 0.8 ± 0.2 0.9 ± 0.2

αB-crystallin pαB-crystrallin-Ser59

1.0 ± 0.2 1.0 ± 0.3

1.1 ± 0.3 1.1 ± 0.3

1.3± 0.4 1.3 ± 0.4

1.1 ± 0.3 1.1 ± 0.3

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590Table 2. Distribution of sHSPs and of psHSPs between fractions at each time point of 591ECC1. Values are the mean (±SEM) percentage of the indicated sHSP or psHSP present in each 592of the three fractions (cytosolic, membrane and cytoskeletal) at the indicated time point, for the 11 593subjects examined. The percentage of the protein in a given fraction was compared across the 594four time points using one way ANOVA with Tukey’s multiple comparison test. * significantly 595different from Pre (P

Small heat shock proteins translocate to the cytoskeleton in human skeletal muscle following eccentric exercise independently of phosphorylation.

Small heat shock proteins (sHSPs) are a subgroup of the highly conserved family of HSPs that are stress inducible and confer resistance to cellular st...
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