Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 1

ARTICLE Hsp25 and Hsp72 content in rat skeletal muscle following controlled shortening and lengthening contractions Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

Andrew M. Holwerda and Marius Locke

Abstract: The cytoprotective proteins, Hsp25 and Hsp72, are increased in skeletal muscle after nondamaging, shortening contractions, but the temporal pattern of expression and stimulatory mechanisms remain unclear. Thus, we sought to define the in vivo temporal patterns of expression for Hsp25 and Hsp72 after 2 opposing contractions types. To do this, male Sprague– Dawley rats had 1 tibialis anterior (TA) muscle electrically stimulated (5 sets of 20 repetitions) while being either forcibly lengthened (LC) or shortened (SC). At 2, 8, 24, 48, 72, or 168 h after the contractions both the stimulated and the nonstimulated (contra-lateral control) TA muscles were removed and processed to examine muscle damage (hemotoxylin and eosin staining) and Hsp content (Western blot analyses). Cross-sections from TA muscles subjected to LCs showed muscle fibre damage at 8 h and thereafter. In contrast, no muscle fibre damage was observed at any time point following SCs. When normalized to contra-lateral controls, Hsp25 and Hsp72 content were significantly (P < 0.01) increased at 24 h (3.1- and 3.8-fold, respectively) and thereafter. There were no significant increases in Hsp25 or Hsp72 content at any time point following SC. These data suggest that LCs, but not SCs, result in Hsp accumulation and that the fibre/cellular damage sustained from LCs may be the stimulus for elevating Hsp content. Key words: concentric, eccentric, Hsp25, Hsp72, muscle contraction, muscle damage. Résumé : La concentration des protéines cytoprotectrices Hsp25 et Hsp72 augmente dans le muscle squelettique a` la suite de contractions avec raccourcissement ne causant pas de lésion, mais le régime temporel de l’expression et les mécanismes de la stimulation ne sont pas bien établis. Cette étude se propose donc de présenter le régime temporel de l’expression in vivo de Hsp25 et Hsp72 a` la suite de deux types opposés de contraction. À cette fin, on stimule électriquement (5 séries de 20 répétitions) le jambier antérieur (« TA ») de rats mâles Sprague–Dawley tout en l’étirant (« LC ») ou le laissant se raccourcir (« SC »). À la 2e, 8e, 24e, 48e, 72e ou 168e heure suivant les contractions, on prélève les TA stimulés et non stimulés (contrôle controlatéral) et on examine les lésions musculaires (coloration a` l’hématoxyline et a` l’éosine) et le contenu en Hsp (transfert Western). Des coupes transversales des TA soumis a` LC révèlent des lésions musculaires a` partir de la 8e heure. Par contre, a` la suite de SC, on n’observe aucune lésion musculaire quel que soit le moment. Après la normalisation par rapport aux valeurs de contrôle, le contenu en Hsp25 et en Hsp72 est significativement plus élevé (P < 0,01) a` la 24e heure (3,1 et 3,8 fois, respectivement) et par après. À la suite de SC, on n’observe aucune augmentation de Hsp25 ou de Hsp72 quel que soit le moment. D’après ces résultats, les LC, mais pas les SC suscitent une accumulation de Hsp et les lésions des fibres musculaires dues aux contractions avec étirement (LC) pourraient être les stimuli a` la source de l’augmentation du contenu en Hsp. [Traduit par le Rédaction] Mots-clés : miométrique, pliométrique, Hsp25, Hsp72, contraction musculaire, lésion musculaire.

Introduction When challenged with protein-damaging or denaturing stressors such as reactive oxygen species or elevated temperatures, cells and tissues respond by synthesizing protective proteins known as heat shock proteins (Hsps) (for reviews see Welch 1992; Noble et al. 2008). While the exact roles of the various Hsps remain to be fully determined, they are thought to allow cells to function during episodes of cellular stress by minimizing perturbations to proteins and hence cellular homeostasis (Welch 1992). Given the myriad of cellular stressors that are imparted on contracting skeletal muscle fibres, it is not surprising that mammalian skeletal muscle Hsp content has been shown to increase following muscle contraction (Ingalls et al. 1998; Milne and Noble 2002; Koh and Escobedo 2004; McArdle et al. 2004; Paulsen et al. 2007; Vissing et al. 2009; Touchberry et al. 2012). As such, understanding the mechanisms of Hsp induction and accumulation in exercised skeletal muscle requires that variables of muscle contraction, such as contraction type, be isolated and examined.

In most instances, skeletal muscle fibres perform shortening contractions (SCs, also known as concentric contractions) where sarcomere length is reduced while overcoming a resistance. In other instances, the resistance imposed on the muscle may be greater than the force capability of the muscle and the “contracting muscle” and its sarcomeres perform lengthening contractions (LC, also known as an eccentric contraction). LCs are performed during daily activities; often where the force of gravity or mass of other objects must be resisted. SCs do not typically result in muscle fibre damage provided the stress from the duration, magnitude, and (or) number of muscle contractions is not excessive. In contrast, LCs are known to result in muscle fibre damage (Nosaka and Clarkson 1995), and are characterized by decreased muscle force (Brooks et al. 1995; Warren et al. 1999), sarcomere disruption (Fridén and Lieber 1992, 2001), a loss of sarcolemma integrity (Fridén et al. 1983; Fridén and Lieber 1992), and intracellular protein loss (Lovering and De Deyne 2004). If severe enough, fibre necrosis results allowing muscle proteins into the circulation (Warren et al. 1993; Lovering and De Deyne 2004).

Received 4 April 2014. Accepted 8 August 2014. A.M. Holwerda and M. Locke. Faculty of Kinesiology and Physical Education, University of Toronto, Toronto, ON M5S 2W6, Canada. Corresponding author: Marius Locke (e-mail: [email protected]). Appl. Physiol. Nutr. Metab. 39: 1–8 (2014) dx.doi.org/10.1139/apnm-2014-0118

Published at www.nrcresearchpress.com/apnm on 26 August 2014.

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

2

Skeletal muscle Hsp content has been shown to increase in mammals following both SCs (Khassaf et al. 2001; Morton 2006; Morton et al. 2009) and LCs (Thompson et al. 2001, 2003; Koh and Escobedo 2004; Paulsen et al. 2007, 2009). However, variations in models such as type (treadmill running or resistance exercise), intensity (treadmill speed and grade or load), duration (time, repetition volume), and subjects used (human or rodent) may result in vastly different stressors to the muscle. Thus, it is challenging to decipher whether increases in Hsp content following muscle contraction results from the indirect effects of elevated temperature or reactive oxygen species or are directly due to muscle damage. Given the differences in stressor(s) imparted on the muscle fibres between SCs and LCs, it seems plausible and possible that magnitude and pattern for Hsp accumulation between the 2 contraction types may also differ. Thus, the purpose of the current study was to define the temporal pattern of muscle Hsp25 and Hsp72 content up to 168 h (7 days) after a bout of 100 controlled LCs or SCs, administered as 5 intermittent sets of 20 contractions. It was hypothesized that both SCs and LCs would elevate muscle Hsp content, but because of the damage sustained from LCs, a greater and more prolonged accumulation would be observed following LCs.

Materials and methods Animals Male Sprague–Dawley rats (N = 65; 364.6 ± 2.1 g, mean ± SE) (Charles River Laboratories, Wilmington, Mass., USA) were housed in pairs and maintained on a constant 12-h light/12-h dark cycle while being fed and provided with water ad libitum. All procedures were approved by the Animal Care Committee at the University of Toronto and were in accordance with the Guidelines for Canadian Council on Animal Care. The surgical and electrical stimulation procedures were performed under anaesthesia (isoflurane/ oxygen gas mixture; 1 L/min). Following the electrically stimulated contractions, animals were monitored for 1 h while they recovered before being returned to the animal care facilities until they were sacrificed under anesthesia. Study design Rats were grouped into either the SC treatment group (n = 30), or the LC treatment group (n = 30). The treatment groups were divided into 6 subgroups representing progressive time points (2, 8, 24, 48, 72, and 168 h) after the contraction bout to develop a temporal pattern of postcontraction muscle Hsp content. Muscles from a separate control group of noncontracted rats (CON) (n = 5) was also used to represent basal Hsp conditions. Stimulation protocol Once anesthetized, animals were positioned in a supine position on a small-animal warming platform (806D, Aurora Scientific Inc., Aurora, Ont., Canada) that was maintained at 37 °C. Hair was removed from the lower right hind limb with natural hair removal cream (Nair, USA). The right knee was secured between 2 vertical stabilizing posts by delicately inserting a 25G × 1.5-inch needle through the hind limb in a lateral orientation, directly distal to the condyles. Two 28G × 0.5-inch needle probe electrodes (Chalgren Enterprises, Calif., USA) were inserted subcutaneously in a longitudinal orientation, directly adjacent to the tibialis anterior (TA) muscle and the leg was manually manipulated for ankle mobility and, more specifically, dorsiflexion. The right paw was secured to the pedal of a computer-controlled servomotor (301C, Aurora Scientific Inc.) with adhesive tape. The pedal was adjusted 3-dimensionally until the secured ankle was in line with the knee and the knee angle was 120°. The pedal was finely adjusted to register zero-torque (g-cm) on the measurement software (DMC, Aurora Scientific Inc.), signifying a neutral and squared leg position.

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

Electrical stimulation was generated from a Grass Stimulator (S88, Grass Technologies, R.I., USA) controlled by a computer hardware interface (604A, Aurora Scientific Inc.) synchronized to the servomotor movements. Stimulation protocols and pedal movements were arranged and executed with computer software (DMC, Aurora Scientific Inc.). Once the rat leg was set in the correct position, optimal stimulation voltage was determined by stimulating the TA muscle to cause dorsiflexion of the paw against a rigid pedal for 0.5-s periods in intervals of 1 V, between 8–12 V with 15 s of rest between stimulations. Isometric torque (g-cm) output was measured and the optimal stimulation voltage for each rat was selected based upon the lowest voltage needed to elicit peak torque. Once peak torque was achieved, the optimal stimulation frequency was determined in a similar manner by keeping optimal voltage constant and increasing the frequency in intervals of 50 Hz (100–300 Hz) with rest periods of 15 s until maximal torque was optimized. Isometric torque data from the single, optimized stimulation was collected and used as the pretreatment peak torque value. In most cases, optimal stimulation parameters were achieved before 5 muscle contractions. Optimal stimulation parameters were typically between 8–10 V and 150 Hz. Prior to each contraction during the treatment protocols, the servomotor passively maneuvered the pedal along with the paw to a position where the ankle angle was 90o for LC or 120o for SC. The difference in starting limb positions for each contraction type allowed for each contraction to occur within the same range of motion. Electrical stimulation during the treatment protocols was initiated for 0.2 s prior to any servomotor movement to allow adequate muscular force development and to collect isometric torque data for each individual contraction. While remaining contracted, servomotor pedal movement was initiated either in the direction of plantar flexion (LC) or dorsiflexion (SC) over a range of 38o in 0.3 s, causing an angular contraction velocity of 127o/s. With this experimental setup, the forced-lengthening of contracted TA muscle in the LC group was isokinetically matched with the loaded shortening contractions in the SC group. Upon the completion of each contraction, stimulation stopped and the pedal passively maneuvered the paw back to the respective starting positions within 3 s. Both LC and SC contraction protocols consisted of 100 stimulated contractions sectioned into 5 sets of 20, with each set separated by 5 min of rest. After the completion of the treatment protocols, post-treatment peak torque was remeasured using a single 0.5-s stimulation against a rigid pedal after 3 min and 10 min. Tissue collection Rats were anaesthetized (isoflurane/oxygen gas mixture; 1 L/min) and sacrificed by exsanguination at various times (2, 8, 24, 48, 72, 168 h) after SCs or LCs. The TA muscles from both the contracted and noncontracted limbs were excised, weighed, and divided into portions for either histochemical or biochemical analyses. Muscle portions used for histochemical analysis were oriented in a cross-sectional manner in OCT mounting gel and rapidly frozen with isopentane that was previously cooled in liquid nitrogen. Portions used for Western blotting were quickly frozen in liquid nitrogen. All samples were stored at –80 °C until processed. Fibre histochemistry Portions of frozen TA muscle in OCT gel were mounted in an American Optical cryostat and cross-sections of 15–20 ␮m were sliced, placed on microscope slides, air dried, and stored at –20 °C. Slides were stained with hematoxylin and eosin (H&E) using standard techniques and examined using a Zeiss Axioplot microscope at 40× magnification to identify muscle fibre damage. Regions of fibre damage were identified by the presence of membrane rupture, swollen cells – indicated by fibre rounding combined with noticeably darker-than-usual staining, “ghost fibres” – indicated Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) Holwerda and Locke

3

Table 1. Tibialis anterior (TA) masses after stimulation treatments.

Shortening contractions 2h 8h 24 h 48 h 72 h 168 h Lengthening contractions 2h 8h 24 h 48 h 72 h 168 h

30 5 5 5 5 5 5 30 5 5 5 5 5 5

Body mass (g)

Stimulated TA muscle (mg)

Nonstimulated TA muscle (mg)

Percent mass change from control (%)

Stimulated muscle mass/body mass (mg/g)

364.4±7.1 362.9±4.5 368.7±5.8 352.1±9.0 363.8±3.7 375.7±8.9

677.0±9.7* 659.6±9.9* 669.2±20.0 633.2±11.7 662.6±15.5 663.6±19.7

636.0±12.9 610.0±35.2 664.2±21.0 633.6±15.7 665.6±12.4 656.6±15.2

106.4 108.1 100.8 99.9 99.5 101.1

1.86±0.06 1.82±0.04 1.81±0.04 1.80±0.02 1.82±0.05 1.77±0.01

362.5±5.1 359.5±3.8 349.2±6.1 366.9±6.0 370.3±8.3 378.5±10.6

680.8±29.4* 683.0±33.0* 685.2±19.2* 691.0±17.7* 672.6±22.1 596.2±11.3*

640.0±22.7 629.6±28.5 635.8±13.7 629.4±12.8 661.6±17.5 660.4±26.2

106.4 108.5 107.8 109.8 101.7 90.3

1.88±0.06 1.90±0.09 1.96±0.03 1.88±0.04 1.82±0.04 1.58±0.04

Note: TA muscles were excised and weighed at the time of sacrifice. Data are expressed in milligrams as mean values of each treatment group (mean ± SE) and in percentage increase/decrease compared with the contralateral nonstimulated muscle.

Table 2. Contraction torque data represented in absolute form. (A) 3 and 10 min postcontraction torque.

Precontractions 3 min postcontractions 10 min postcontractions

Shortening contractions

Lengthening contractions

304.9±6.36 193.0±7.9 209.0±8.81

269.6±6.57 89.2±4.7 88.3±5.6

Fig. 1. Isometric torque is decreased following both shortening (SC) and lengthening (LC) contractions. Isometric torque measured at 3 min and 10 min after the completion of the LC and SC bouts. Data are expressed as values normalized to precontraction torque measurements (mean ± SE; n = 30 in each contraction type). *, P < 0.001 compared with SC within time point; †, P < 0.001 compared with rest. Pre–post, pre-/postcontraction.

100

(B) First and twentieth torque measurements for each contraction set. Contraction bout

1st rep

20th rep

1st rep

20th rep

SET1 SET2 SET3 SET4 SET5

292.0±13.5 260.7±11.6 234.2±12.8 215.4±13.1 202.1±11.6

202.2±10.5 205.6±8.4 183.0±8.4 167.1±8.4 156.6±8.0

272.4±8.0 241.5±5.5 164.6±6.9 124.0±7.7 82.3±5.8

171.3±7.6 115.0±6.0 78.0±5.7 57.4±5.0 42.5±4.6

Note: The mean values for torque measurements used in Tables 1, 2A, and 2B expressed as absolute values (g-cm; mean ± SE). The table expresses mean values for the isometric contractions performed pre- and postcontraction bout (3 and 10 min) and during the isometric phase prior to servomotor pedal movement before to each individual contraction throughout the bout (SET1–SET5). The data were not statistically compared in this table because of the between-treatment differences of initial muscle length before contraction.

by a missing fibre surrounded by otherwise healthy-looking fibres, and mononuclear cell infiltration – indicated by swarming pattern of dark purple cells. Once locations of fibre damage were identified, magnification was increased to 400× and images were captured to represent the histopathology within these areas. Protein determination and Western blot analyses Frozen portions of TA muscle (150–300 mg) were homogenized in 10 volumes of 600 mmol/L NaCl and 15 mmol/L Tris (pH 7.5) at 4 °C using an Ultra-Turrax T8 grinder (IKA Labortechnik, Staufen, Germany). Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Based on the determined protein concentrations, sample volumes containing 250 ␮g of protein were loaded into 5%–15% gradient acrylamide gels with 1 lane containing purified Hsp25 and Hsp72. Protein samples were separated using 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis according to the method described by Laemmli. (1970). Separated proteins were transferred from the gel slab to nitrocellulose membranes (0.22 ␮m pore size, Bio-Rad Laboratories, Mississauga, Ont., Canada) using the method of Towbin et al. (1979), and modified to the Bio-Rad mini-protean II gel transfer system as described by Frier et al. (2008). Nitrocellulose membranes were blocked with 5% w/v nonfat dried milk power (NFDM) dissolved in Tris-buffered saline

Percentage of pre–peak torque (%)

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

n

80

60

*

*

LC SC

40

20

0

3min

10min

(TBS) for 1 h at room temperature. Blocked membranes were washed twice for 5 min each with TBS plus Tween (TTBS) before being incubated overnight at 4 °C with a polyclonal antibody specific for Hsp25 (ADI-SPP-715, ENZO, USA) or Hsp72 (ADI-SPA-812, ENZO, USA) diluted 1:1000 in TTBS with 2% NFDM. Incubated blots were washed twice in TTBS for 5 min. Membranes were incubated for 1 h at room temperature with goat anti-rabbit secondary antibody conjugated to horse-radish peroxidase (70745, Cell Signaling Technology, USA) in a 1:2500 dilution in TTBS and 2% NFDM. Membranes were washed twice in TTBS and once in TBS for 5 min each before being treated with a luminol-based solution (Luminato Forte, Millipore, USA), exposed to film (CLM5810, Bioflex, USA), and developed. Developed films were digitally scanned at 1200DPI and band densities representing Hsp25 or Hsp72 content were quantified using ImageJ software (version 1.43). Particular to Hsp25, both bands were quantified and counted as total Hsp25 content. Values obtained from scanned bands were compared with the contralateral, noncontracted muscles and Hsp quantities were represented as fold-change from the contralateral muscle. Hsp25 and Hsp72 in both TA muscle samples taken from Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 4

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

Fig. 2. (A) Isometric torque is decreased after the each contraction type. Isometric torque measured during the twentieth repetition of each set was normalized to isometric torque measured during the initial contraction for each subject. (B) Decreased isometric torque between consecutive sets for each contraction type. Mean decrease in relative isometric torque between each set of contractions for lengthening (LC) and shortening (SC) contractions. Data are presented as means ± SE and are expressed as percentage decline. *, P < 0.001 compared with SC.

A

SET1

SET2

SET3

SET4

SET5

B

-10 -20

Intra-set Torque Decrease (%)

Torque Decrease from 1st REP (%)

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

0

-30 -40 -50

*

-60 -70 -80 -90

* LC SC

* *

the group of completely noncontracted control rats was also quantified by Western blotting and expressed as fold-change from one another, representing basal Hsp content. Creatine kinase assay As a marker of skeletal muscle damage, creatine kinase content was assessed using an enzyme-linked immunosorbent assay (ELISA) using a Coulter microplate reader. Kits from Abcam (cat. no. ab 187396) were used to determine the amount of creatine kinase in a known volume of rat serum according to the manufactures protocol. Standard curves were constructed and creatine kinase content determined from values obtained by an end point reaction at 450 nm. Statistics A 2-way ANOVA with independent measures was used to compare the interaction effect of time versus contraction type for Hsp25 and Hsp72. A 2-way ANOVA with repeated measures was used to compare the interaction effect of time versus torque for all contraction torque data, except inter-set decreases in torque, in which a paired t test was used. A Bonferroni post hoc test was used to detect differences when the ANOVA revealed a significant interaction. All data are reported as means ± SE. The level of significance for all statistical tests was set at P < 0.05.

Results Muscle mass To assess whether the 2 contraction types resulted in changes in muscle mass because of swelling or edema, muscle mass was measured at the time of sacrifice (see Table 1). When SC and nonstimulated muscle masses were compared, slight but significant increases (P < 0.05) in muscle mass were detected at 2 h (6.4%) and 8 h (8.1%) but not thereafter. After LCs, significant increases (P < 0.05) in muscle mass between stimulated and nonstimulated muscles were detected at 2 h (6.4%), 8 h (8.5%), 24 h (7.8%), and 48 h (9.8%). At 72 h, muscle mass between LC muscles and contralateral muscles was similar but by 168 h the mass of the LC-stimulated muscles was decreased (–9.7%) compared with contralateral controls. By expressing the ratio of stimulated TA muscle mass by the total body mass (Table 1), comparisons between contraction types at each time point could be made. No significance at any time point (P > 0.05) was detected when comparing muscle mass after SCs to muscle mass after LCs. As a whole, these data suggest that while both SCs and LCs showed initial elevations in muscle mass, the mass of SC muscles returned to precontraction levels within a few hours while LCs resulted in a sustained

0

-5

LC SC

-10

-15

*

*

elevation of muscle mass that was followed by a drop in muscle mass. Contractile torque To gain insight into how each contraction type influenced muscle function, peak torque (g-cm) was measured before the contraction bouts and at 3 min and 10 min after the 100 contractions (Table 2). The postcontraction peak torque data are also represented as a percentage relative to the precontraction peak torque values for each animal (Fig. 1). A significant decrease in peak torque was detected at 3 min (LC: 33.3% ± 9.1%; SC: 63.2% ± 11.7% of precontraction values; P < 0.05) and 10 min (LC: 33.1% ± 11.2%; SC: 68.4% ± 12.9% of precontraction values; P < 0.05) after both contraction types. However, the decrease in peak torque after the LCs was significantly greater when compared with SCs at both time points (P < 0.05), suggesting that there was an alternate mechanism causing contractile impairment with LCs. Isometric torque (g-cm) measurements from the first and twentieth contraction of each completed set are presented in Table 2. Additionally, isometric torque measured in the twentieth repetition of each set was also normalized to the isometric torque measured in the first repetition of the first set (Fig. 2A). A declining pattern of isometric torque from each set for both contraction types was observed. However, for each set the percentage decline in torque was significantly (P < 0.05) greater following LCs when compared with SCs. In addition, the 5 sets of LCs caused a significantly (P < 0.05) more rapid decline in isometric torque between each progressive set when compared with SCs (LC: –11.7% ± 0.78%; SC: –4.7% ± 0.60%) (Fig. 2B). Taken together, these data indicate that LCs caused a more sustained impairment in contractile function than SCs. This decreased occurred as early as the first set and continued throughout the entire contraction bout. Muscle fibre morphology H&E staining was used to visualize TA muscle fibre morphology at each time point after both contraction types (Fig. 3). Two hours after each contraction type no changes in morphology were detected (Fig. 3A and 3B) and the morphology was similar to that observed for controls (data not shown). At 8 h after LCs, muscle fibres showed evidence of damage in the form of swollen fibres, vacant or ghost fibres, ruptured endomysium, as well as infiltration by mononuclear cells (Fig. 3C). This pattern of fibre damage after LCs continued up to 72 h (Fig. 3C, 3E, 3G, and 3I). In most cases, TA muscle fibres were repaired and intact by 168 h (Fig. 3K) after LCs and at this time point muscle morphology was similar to the SC muscles and controls. However, some mononuclear cells Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) Holwerda and Locke

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

Fig. 3. Muscle cross-sections show damage after lengthening contractions (LCs). Hemotoxylin and eosin-stained tibialis anterior (TA) cross- sections displaying visual muscle fibre damage. (A, C, E, G, I, K) Cross-sections of TA muscles after LCs; (B, D, F, H, J, L) crosssections of TA muscles after shortening contractions (SCs). Visible fibre damage is present at 8 h after LC (C) along with a noticeable increase in deep purple cells disseminating around the damaged fibres. No visible fibre damage or inflammatory cell infiltration was observed after SC. Magnification is 100×.

5

tent in TA muscles from nonstimulated muscles (CON). After the LCs, although elevated more than 2-fold at 24 h (2.2 ± 0.38-fold) and 72 h (2.5 ± 0.49-fold), no statistical difference was detected when compared with CON. At 48 h (3.1 ± 0.53-fold increase) and at 168 h (3.0 ± 0.83-fold increase) (Fig. 4A). When the muscle Hsp25 content of the 2 contraction types was compared at the specific time points, significant (P < 0.05) elevations in Hsp25 were detected between LCs and SCs at 8 h and all points thereafter. In contrast with the elevated Hsp25 observed after LCs, no significant increases in Hsp25 content was observed at any time point following SCs. Muscle Hsp72 content was also expressed relative to the contralateral control TA muscle (Fig. 4B). When compared with CON, muscle Hsp72 content was significantly (P < 0.05) elevated at 24, 48, and 72 h by 3.8 ± 0.66-, 2.6 ± 0.49-, and 3.22 ± 0.57-fold, respectively. No increase in muscle Hsp72 content was observed after SCs for any of the time points examined. When Hsp72 content between contraction types within individual time points was compared, a significant elevation (P < 0.05) was detected at 8 h (2.3 ± 0.42-fold) and thereafter. Taken together, these data suggest that electrically stimulated 100 LCs are capable of increasing muscle Hsp content while 100 SCs does not result in an elevation of muscle Hsp25 or Hsp72 content. Serum creatine kinase content following muscle contractions As a marker of muscle damage, serum creatine kinase was assessed by the ELISA technique (Fig. 5). When compared with serum creatine kinase content from controls (no muscle contractions), there was an approximately 2-fold increase in serum creatine kinase content at 2 h after SCs and at 8 and 24 h after LCs. However, despite these increases no statistically significant differences were detected between groups or from controls.

Discussion

were still detected around regenerating muscle fibres at 168 h (Fig. 3K) post-LC. In contrast to the damage and disruption observed in muscle fibres following LCs, there was no aberrant fibre morphology observed in muscles after SCs at any time point (Fig. 3B, 3D, 3F, 3H, 3J, and 3L). These observations indicate that LCs resulted in muscle damage while SCs did not result in any detectable muscle damage. Muscle Hsp25 and Hsp72 protein content after muscle contractions Hsp25 and Hsp72 were detected in all TA muscles examined by Western blot analyses. Following quantification by densitometry, muscle Hsp content was expressed relative to the Hsp content in the nonstimulated (contra-lateral) muscle of the same animal. A baseline or CON Hsp content was determined from the Hsp con-

The content of Hsp25 and Hsp72 was examined in the rat TA muscle after 100 electrically stimulated shortening (nondamaging) or lengthening (damaging) contractions using a controlled in vivo model of muscle contraction. As expected, forced-LCs caused a significant decrease in contractile function and an appreciable amount of muscle fibre damage. These changes in muscle function and morphology were followed within hours by an accumulation of Hsp25 and Hsp72 content, which remained elevated at 7 days. Although SCs also resulted in a significant decline in contractile function, neither muscle fibre damage nor elevation of Hsp content was observed. While the exact reason for the divergent pattern of Hsp25 and Hsp72 expression observed between LCs and SCs cannot be ascertained from the present study, a likely explanation is that the elevated Hsp content stems from the muscle fibre damage that follows LCs. Since it is well known that damaged or denatured proteins are a major inducer of Hsps (Welch 1992), it follows that a greater Hsp content would be expected following LCs. A significant decrease in torque is typically indicative of contraction-induced muscle damage and (or) fatigue. In the present investigation, the 100 SCs performed resulted in significantly reduced muscle contractile function yet no elevation of muscle Hsp content occurred. It should be noted that the SCs used were isokinetically matched to the LCs, thus controlling for contraction velocity, time contracted, and mechanical work. Although similar declines in isometric torque were observed after the first set for both LCs and SCs (–38% for LC and –31% for SC), the patterns for torque decline differed thereafter, revealing that LCs caused a greater decline in torque between each consecutive set (SC: –4.5% vs. LC: –11.7%). Furthermore, muscles subjected to SCs appeared to plateau after the first set while muscles subjected to LCs continued to decline. Although the exact reason(s) for these differences cannot be determined from the present study, the most likely Published by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 6

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

Fig. 4. (A) Muscle Hsp25 and Hsp72 content are elevated after lengthening (LC) but not shortening (SC) contractions. Hsp25 protein content at 2, 8, 24, 48, 72, and 168 h after the LC and SC bout (expressed as fold-change from values measured in contralateral control muscle). (B) Hsp72 protein content measured at 2, 8, 24, 48, 72, and 168 h after the LC and SC bout (expressed as fold-change from values measured in contralateral control muscle). Values are means ± SE; n = 5 in each contraction type. *, P < 0.05 compared with SC within time point; †, P < 0.05 compared with control (con) group. Representative blots showing only contracted samples were electrophoresed with purified Hsp25 or Hsp72.

A

B

Hsp25

*

*

2 1

4

2h

8h

24h

48h

72h

(3days)

*

LC SC

*

3

*

*

2 1 0

con

168h

(7days)

LC

con

2h

8h

24h

48h

72h

168h

(3days)

(7days)

EC LC CC SC

SC

con

con

2h

8h

24h

48h

72h 168h +con (3days)

(7days)

Fig. 5. Serum creatine kinase content after muscle contractions. Creatine kinase content was assessed using an enzyme-linked immunosorbent assay as described in the Materials and methods section. Serum was measured at 2, 8, 24, 48, 72, and 168 h after either shortening or lengthening muscle contractions. Data are expressed as values normalized to controls with no tibialis anterior muscle contractions (mean ± SE; n = 5 in each contraction type). No significant differences were detected. Lengthening contractions are represented by open bars while shortening contractions are solid bars.

Lengthening Contracons Shortening Contracons

250 200 150 100 50 2

* Fold chang change a e (from control contr t ol leg)

Fold change (from control limb)

*

3

300

Hsp72

5

*

LC LC SC SC con

4

0

Creatine Kinase Content (% of Control)

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

5

8 24 48 72 Hours Following Contraction

168

explanation for these differences are that the changes in contractile function following SCs are due to metabolic alterations (Smith and Newham 2006). Additional support for the notion that the drop in tetanic force after SCs is likely due to a metabolically related fatigue, which can be gained by the greater recovery of tetanic tension that was noted at 3 and 10 min following SCs compared with LCs. Although nondamaging endurance-type exercise can induce the accumulation of muscle Hsps (Khassaf et al. 2001; Morton 2006; Morton et al. 2009), the intensity, duration, and a continual nature of nondamaging muscle contractions (endurance exercise) may need to reach a threshold to cause sufficient protein perturbation to elevate Hsp content. This relationship between exercise intensity and expression of muscle Hsps

con

2h

2h

8h

8h

1d

24h

2d

48h

3d

7d

+con

72h 168h +con (3days)

(7days)

was demonstrated by Milne and Noble (2002), who reported that the accumulation of Hsps after nondamaging exercise is influenced by increased treadmill running speeds. Thus, it is likely that a sufficient level of stress in the form of temperature or reactive oxygen species must be generated from SCs to induce Hsps expression. Given the role and protective nature of the Hsps, the findings that nondamaging muscle contractions do not sufficiently induce Hsp accumulation may have implications for exercise training, muscle adaptation, and muscle rehabilitation. In contrast with SCs, the drop in tetanic force following the 100 stimulated LCs was more severe and longer lasting. The likely cause of this lasting drop was cellular damage coupled with impaired excitation–contraction coupling (Faulkner et al. 1989; Warren et al. 1993; Brooks et al. 1995; Febbraio et al. 2002, 2004). With regards to structural protein damage, vital cytoskeletalanchoring proteins such as desmin and dystrophin are known to be disrupted during or following LCs (Barash et al. 2002; Koh and Escobedo 2004). In the present study, muscle fibre damage was noted after LCs, strongly suggesting that perturbations to contractile proteins occurred. It follows that these disturbances to proteins and hence muscle fibre structure elicited the accumulation of Hsp25 and Hsp72. Previous work by Ingalls et al. (1998) has demonstrated a relationship between increased muscle Hsp72 content and decreasing contractile proteins following damaging LCs. Whether the damage to these or other proteins is the stimulus for an elevation of Hsps remains to be determined. Another major difference between SCs and LCs was the infiltration of mononuclear or immune cells (Fig. 4). When muscle fibres are stressed and proteins are damaged, Hsps are synthesized in an attempt to restore homeostasis and ultimately survive. However, some fibres with an elevated Hsp content will not survive and will undergo necrosis. This allows proteins, including Hsps, to be released into the extracellular environment where they are capable of promoting immune and (or) inflammatory responses by serving as a signal for cell damage. Indeed, released proteins, termed “alarmins” or Damage Associated Molecular Patterns can initiate a ligand-receptor mediated immune and (or) inflammatory response. Thus, there is the potential for extracellular Hsps to act as alarmins and communicate with immune cells to initiate the inflammatory response (Asea et al. 2000; Wallin et al. 2002). The respective origin and interactions of extracellular Hsps in damPublished by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI)

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

Holwerda and Locke

aged muscle fibres is currently unknown and hence the exact role that Hsp and other proteins play in these processes remains to be determined. The cellular protection afforded by increased Hsps has been demonstrated using transgenic mice overexpressing Hsp70 and other means. Less of a reduction in contractile force was demonstrated after contraction-induced damage in the extensor digitorum longus muscle when compared with wild-type mice (McArdle et al. 2004). More recently, an increased muscle Hsp content elicited by a prior heat shock appeared to enhance muscle reconditioning after damaging treadmill running (Touchberry et al. 2012). The investigators reported lower plasma markers of muscle damage along with increased total muscle protein concentration and myosin heavy-chain protein content. In response to fibre damage, both Hsp25 and Hsp72 appear to increase in concentration within the muscle and translocate to the myofibrils after contractioninduced damage (Lavoie et al. 1993; Koh 2002; Paulsen et al. 2007, 2009; Vissing et al. 2009). Whether Hsps are involved in attempting to stabilize the sarcomere remains to be determined but it does suggest that Hsps may aid in protection, repair, and possibly muscle growth. Indeed, an increased Hsp content from LCs or otherwise may benefit muscle adaptation, and thus may have a therapeutic application for at-risk populations (i.e., frail, dystrophic, etc.). It may be that an elevated muscle Hsp content from LCs or otherwise, may contribute to more rapid adaptation thus minimizing the effect of future damaging muscle contractions – otherwise known as the repeated-bout effect (RBE) (McHugh 2003). At present, the only known method of minimizing the soreness, force decrement, and cellular damage from LCs is a prior, less severe bout of LCs/exercise. Hsp have been proposed as a potential mechanism by which the RBE may minimize the damage associated with LCs (McHugh 2003). In conclusion, controlled LCs of the rat TA muscle caused a marked decrease in contractile function and muscle fibre damage while inducing a robust and lasting increase in muscle Hsp25 and Hsp72 content. In contrast, isokinetically matched SCs caused a significant decline in contractile function, but did not elevate Hsp25 or Hsp72 content. Thus, it appears that the stimulus for Hsp induction from intermittent muscle contractions (i.e., resistance exercise) may occur from resultant fibre damage rather than metabolically fatiguing, nondamaging muscle contractions.

References Asea, A., Kraeft, S.K., Kurt-Jones, E.A., Stevenson, M.A., Chen, L.B., Finberg, R.W., et al. 2000. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6(4): 435–442. doi:10.1038/74697. PMID:10742151. Barash, I.A., Peters, D., Fridén, J., Lutz, G.J., and Lieber, R.L. 2002. Desmin cytoskeletal modifications after a bout of eccentric exercise in the rat. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283(4): R958–R963. PMID:12228066. Brooks, S.V., Zerba, E., and Faulkner, J.A. 1995. Injury to muscle fibres after single stretches of passive and maximally stimulated muscles in mice. J. Physiol. 488(Pt 2): 459–469. PMID:8568684. Faulkner, J.A., Jones, D.A., and Round, J.M. 1989. Injury to skeletal muscles of mice by forced lengthening during contractions. Q. J. Exp. Physiol. 74(5): 661–670. PMID:2594927. Febbraio, M.A., Steensberg, A., Walsh, R., Koukoulas, I., van Hall, G., Saltin, B., and Pedersen, B.K. 2002. Reduced glycogen availability is associated with an elevation in HSP72 in contracting human skeletal muscle. J. Physiol. 538(Pt 3): 911–917. doi:10.1113/jphysiol.2001.013145. PMID:2290094. Febbraio, M.A., Mesa, J.L., Chung, J., Steensberg, A., Keller, C., Nielsen, H.B., et al. 2004. Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans. Cell Stress Chaperones, 9(4): 390–396. doi:10.1379/CSC-24R1.1. PMID:15633297. Fridén, J., and Lieber, R.L. 1992. Structural and mechanical basis of exerciseinduced muscle injury. Med. Sci. Sports Exercise, 24(5): 521–530. PMID: 1569848. Fridén, J., and Lieber, R.L. 2001. Eccentric exercise-induced injuries to contractile and cytoskeletal muscle fibre components. Acta Physiol. Scand. 171(3): 321– 326. doi:10.1046/j.1365-201x.2001.00834.x. PMID:11412144. Fridén, J., Sjöström, M., and Ekblom, B. 1983. Myofibrillar damage following intense eccentric exercise in man. Int. J. Sports Med. 4(03): 170–176. doi:10. 1055/s-2008-1026030. PMID:6629599.

7

Frier, B.C., Noble, E.G., and Locke, M. 2008. Diabetes-induced atrophy is associated with a muscle-specific alteration in NF-␬B activation and expression. Cell Stress Chaperones, 13(3): 287–296. doi:10.1007/s12192-008-0062-0. PMID: 18633731. Ingalls, C.P., Warren, G.L., and Armstrong, R.B. 1998. Dissociation of force production from MHC and actin contents in muscles injured by eccentric contractions. J. Muscle Res. Cell Motil. 19(3): 215–224. doi:10.1023/A:1005368831198. PMID:9583362. Khassaf, M., Child, R.B., McArdle, A., Brodie, D.A., Esanu, C., and Jackson, M.J. 2001. Time course of responses of human skeletal muscle to oxidative stress induced by nondamaging exercise. J. Appl. Physiol. 90(3): 1031–1035. PMID: 11181616. Koh, T.J. 2002. Do small heat shock proteins protect skeletal muscle from injury? Exercise Sport. Sci. Rev. 30(3): 117–121. doi:10.1097/00003677-200207000-00005. Koh, T.J., and Escobedo, J. 2004. Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions. Am. J. Physiol. Cell Physiol. 286(3): C713–C722. doi:10.1152/ajpcell.00341.2003.PMID: 14627610. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259): 680–685. doi:10.1038/227680a0. PMID:5432063. Lavoie, J., Gingras-Breton, G., Tanguay, R., and Landry, J. 1993. Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. J. Biol. Chem. 268(5): 3420–3429. PMID:8429018. Lovering, R.M., and De Deyne, P.G. 2004. Contractile function, sarcolemma integrity, and the loss of dystrophin after skeletal muscle eccentric contractioninduced injury. Am. J. Physiol. Cell Physiol. 286(2): 230C–238C. doi:10.1152/ajpcell. 00199.2003. PMID:14522817. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193(1): 265–275. PMID: 14907713. McArdle, A., Dillmann, W.H., Mestril, R., Faulkner, J.A., and Jackson, M.J. 2004. Overexpression of HSP70 in mouse skeletal muscle protects against muscle damage and age-related muscle dysfunction. FASEB J. 18: 355–357. doi:10. 1096/fj.05-4935fje. PMID:14688209. McHugh, M.P. 2003. Recent advances in the understanding of the repeated bout effect: the protective effect against muscle damage from a single bout of eccentric exercise. Scand. J. Med. Sci. Sports, 13(2): 88–97. doi:10.1034/j.16000838.2003.02477.x. PMID:12641640. Milne, K.J., and Noble, E.G. 2002. Exercise-induced elevation of HSP70 is intensity dependent. J. Appl. Physiol. 93(2): 561–568. doi:10.1152/japplphysiol. 00528.2001. PMID:12133865. Morton, J.P. 2006. Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise. J. Appl. Physiol. 101(1): 176–182. doi:10.1152/jappl physiol.00046.2006. PMID:16565353. Morton, J.P., Croft, L., Bartlett, J.D., MacLaren, D.P.M., Reilly, T., Evans, L., et al. 2009. Reduced carbohydrate availability does not modulate training-induced heat shock protein adaptations but does upregulate oxidative enzyme activity in human skeletal muscle. J. Appl. Physiol. 106(5): 1513–1521. doi:10.1152/ japplphysiol.00003.2009. PMID:19265068. Noble, E.G., Milne, K.J., and Melling, C.W.J. 2008. Heat shock proteins and exercise: a primer. Appl. Physiol. Nutr. Metab. 33(5): 1050–1065. doi:10.1139/H08069. PMID:18923583. Nosaka, K., and Clarkson, P.M. 1995. Muscle damage following repeated bouts of high force eccentric exercise. Med. Sci. Sports Exercise, 27(9): 1263–1269. doi:10.1249/00005768-199509000-00005. PMID:8531624. Paulsen, G., Vissing, K., Kalhovde, J.M., Ugelstad, I., Bayer, M.L., Kadi, F., et al. 2007. Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293(2): R844–R853. doi:10.1152/ ajpregu.00677.2006. Paulsen, G., Lauritzen, F., Bayer, M.L., Kalhovde, J.M., Ugelstad, I., Owe, S.G., et al. 2009. Subcellular movement and expression of HSP27, ␣B-crystallin, and HSP70 after two bouts of eccentric exercise in humans. J. Appl. Physiol. 107(2): 570–582. doi:10.1152/japplphysiol.00209.2009. PMID:19498098. Smith, I.C.H., and Newham, D.J. 2006. Fatigue and functional performance of human biceps muscle following concentric or eccentric contractions. J. Appl. Physiol. 102(1): 207–213. doi:10.1152/japplphysiol.00571.2006. PMID:16990506. Thompson, H.S., Scordilis, S.P., Clarkson, P.M., and Lohrer, W.A. 2001. A single bout of eccentric exercise increases HSP27 and HSC/HSP70 in human skeletal muscle. Acta Physiol. Scand. 171(2): 187–193. doi:10.1046/j.1365-201x.2001. 00795.x. PMID:11350279. Thompson, H.S., Maynard, E.B., Morales, E.R., and Scordilis, S.P. 2003. Exerciseinduced HSP27, HSP70 and MAPK responses in human skeletal muscle. Acta Physiol. Scand. 178(1): 61–72. doi:10.1046/j.1365-201X.2003.01112.x. PMID: 12713516. Touchberry, C.D., Gupte, A.A., Bomhoff, G.L., Graham, Z.A., Geiger, P.C., and Gallagher, P.M. 2012. Acute heat stress prior to downhill running may enhance skeletal muscle remodeling. Cell Stress Chaperones, 17(6): 693–705. doi:10.1007/s12192-012-0343-5. PMID:22589083. Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proPublished by NRC Research Press

Pagination not final (cite DOI) / Pagination provisoire (citer le DOI) 8

Warren, G.L., Hayes, D.A., Lowe, D.A., and Armstrong, R.B. 1993. Mechanical factors in the initiation of eccentric contraction-induced injury in rat soleus muscle. J. Physiol. 464: 457–475. PMID:8229813. Warren, G.L., Lowe, D.A., and Armstrong, R.B. 1999. Measurement tools used in the study of eccentric contraction-induced injury. Sports Med. 27(1): 43–59. doi:10.2165/00007256-199927010-00004. PMID:10028132. Welch, W.J. 1992. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72(4): 1063–1081. PMID:1438579.

Appl. Physiol. Nutr. Metab. Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Irvine on 11/07/14 For personal use only.

teins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76(9): 4350–4354. doi:10.1073/pnas. 76.9.4350. PMID:388439. Vissing, K., Bayer, M.L., Overgaard, K., Schjerling, P., and Raastad, T. 2009. Heat shock protein translocation and expression response is attenuated in response to repeated eccentric exercise. Acta Physiol. 196(3): 283–293. doi:10. 1111/j.1748-1716.2008.01940.x. Wallin, R.P.A., Lundqvist, A., Moré, S.H., von, Bonin, A., Kiessling, R., and Ljunggren, H.-G. 2002. Heat-shock proteins as activators of the innate immune system. Trends Immunol. 23(3): 130–135. doi:10.1016/S1471-4906(01) 02168-8. PMID:11864840.

Appl. Physiol. Nutr. Metab. Vol. 39, 2014

Published by NRC Research Press