Reduced sarcoplasmic reticulum content of releasable Ca2+

in rat soleus muscle fibres after eccentric contractions

J. S. Nielsen,1 K. Sahlin1,2,3 and N. Ørtenblad1

1 Institute of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark

2 Stockholm University College of Physical education and Sports, Stockholm, Sweden

3 Department of Physiology and Pharmacology, Karolinska Intitutet, Stockholm, Sweden

Received 4 April 2007,

revision requested 19 April 2007,

final revision received 22 May

2007,

accepted 26 May 2007

Correspondence: N. Ørtenblad,

Institute of Sports Science and

Clinical Biomechanics, University

of Southern Denmark, Campusvej

55, 5230 Odense M, Denmark.

E-mail: nortenblad@health.sdu.dk

Abstract

Aim: The purpose was to evaluate the effects of fatiguing eccentric con-

tractions (EC) on calcium (Ca2+) handling properties in mammalian type I

muscles. We hypothesized that EC reduces both endogenous sarcoplasmic

reticulum (SR) content of releasable Ca2+ (eSRCa2+) and myofibrillar Ca2+

sensitivity.

Methods: Isolated rat soleus muscles performed 30 EC bouts. Single fibres

were isolated from the muscle and after mechanical removal of sarcolemma

used to measure eSRCa2+, rate of SR Ca2+ loading and myofibrillar Ca2+

sensitivity.

Results: Following EC maximal force in whole muscle was reduced by 30%

and 16/100 Hz force ratio by 33%. The eSRCa2+ in fibres from non-stimulated

muscles was 45 � 5% of the maximal loading capacity. After EC, eSRCa2+ per

fibre CSA decreased by 38% (P = 0.05), and the maximal capacity of SR Ca2+

loading was depressed by 32%. There were no effects of EC on either myofi-

brillar Ca2+ sensitivity, maximal Ca2+ activated force per cross-sectional area

and rate of SR Ca2+ loading, or in SR vesicle Ca2+ uptake and release.

Conclusions: We conclude that EC reduces endogenous SR content of re-

leasable Ca2+ but that myofibrillar Ca2+ sensitivity and SR vesicle Ca2+

kinetics remain unchanged. The present data suggest that the long-lasting

fatigue induced by EC, which was more pronounced at low frequencies (low

frequency fatigue), is caused by reduced Ca2+ release occurring secondary to

reduced SR content of releasable Ca2+.

Keywords calcium, eccentric contraction, Sarcoplasmic reticulum.

Calcium (Ca2+) handling is a major determinant of both

skeletal muscle force generation [sarcoplasmic reticu-

lum (SR) Ca2+ release and myofibrillar Ca2+ sensitivity]

and force relaxation (SR Ca2+ uptake) (Westerblad &

Allen 2002, Steele & Duke 2003). The rate of SR Ca2+

release (measured in vitro) is reduced in rat muscle

stimulated to fatigue (Ørtenblad et al. 2000), and in

human muscle after whole body exercise to fatigue

(Leppik et al. 2004). Reduction in SR Ca2+ release likely

explains the reduction in Cytosolic [Ca2+] ([Ca2+]i)

observed during tetanic contraction to fatigue in single

fibres (Westerblad et al. 1993). A reduction in Ca2+

release may be a consequence of disturbances of the

process of Ca2+ release or of reduced endogenous SR

content of releasable Ca2 (eSRCa2+).

During in vitro intense stimulation of the muscle, the

total content of Ca2+ increases, possibly buffered by the

SR, which leads to irreversible muscle damage (Gissel &

Clausen 2000). However, with less intense stimulations

the free Ca2+ in the SR lumen ([Ca2+]SR) measured

in vivo, is reduced by 50 lm during a single twitch

elicited through nerve stimulation (Rudolf et al. 2006).

Additionally, approximately 40% of the SR Ca2+

content is released during a single 40 Hz tetanus

(1.2 s) in frog fibres (Somlyo et al. 1981) and when

the SR Ca2+ reuptake is blocked, 4–8 single action

Acta Physiol 2007, 191, 217–228

� 2007 The AuthorsJournal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x 217

potentials are sufficient to deplete SR Ca2+ (Posterino &

Lamb 2003), emphasizing that SR Ca2+ stores can be

limited. The hypothesis that eSRCa2+ is reduced in

fatigue is supported by studies in intact single fibres

from cane toad (Kabbara & Allen 1999). When fibres

were stimulated to fatigue (50% of initial tension), the

rapidly releasable Ca2+ from SR fell to 46% of control.

However, in intact fibres it is not possible to differen-

tiate between the effects of reduced eSRCa2+ content

per se and the effects of changes in the intracellular

milieu (i.e. H+ Pi, ATP, Mg2+) on the release process or

buffering of Ca2+. Furthermore, the composition of the

bulk solution surrounding the single fibre can be quite

different from that in the interstitial fluid in intact

muscle and thus influence the intracellular ionic com-

position during contraction. In mechanically skinned

single fibre, the sarcolemma is mechanically removed.

This gives direct access to the intracellular milieu, and

allows selective modulation of the milieu. The mechan-

ically skinned single fibre technique has been used to

estimate eSRCa2+ content in fibres from resting muscles

(Trinh & Lamb 2006) but the effect of fatiguing

exercise on eSRCa2+ has not previously been investi-

gated.

Inorganic phosphate (Pi) may increase 5- to 10-fold in

fatigued muscle, due to degradation of creatine phos-

phate. It has been hypothesized that Pi moves into SR

leading to a precipitation of Ca2+-phosphate (Posterino

& Fryer 1998). This will decrease the releasable amount

of Ca2+, reducing both the rate and amount of Ca2+

released from SR. There is some experimental support

for the hypothesis, in that experimental increases of Pi

have been shown to reduce tetanic [Ca2+]i in both intact

(Westerblad & Allen 1996b) and skinned fibres (Dutka

et al. 2005). Furthermore, there is evidence from both

studies in rat (Ørtenblad et al. 2000) and humans

(Booth et al. 1997) that the rate of SR Ca2+ uptake

(measured in vitro) is reduced in fatigued muscle.

A delayed removal of [Ca2+]i has also been observed

in situ in fatigued single fibres (Westerblad & Allen

1996a). A reduced rate of SR Ca2+ uptake will act

additive to Ca2+-phosphate precipitation and may

further reduce SR luminal [Ca2+]. A decrease in releas-

able SR Ca2+ may play an important role in fatigue,

however little is known about the effects of fatigue on

SR luminal releasable Ca2+ in intact mammalian mus-

cle.

Fatigue evoked by eccentric contractions (EC) is often

characterized by a more pronounced reduction at low,

than at high stimulation frequencies and has been

denoted low frequency of fatigue (LFF) (Edwards et al.

1977). LFF may persist for several days and is more

pronounced after EC than after concentric and isomet-

ric contractions (Jones et al. 1989). Accordingly, LFF is

not related to acute changes in metabolic status but may

instead be caused by disturbed Ca2+ handling (Ingalls

et al. 1998). During LFF, the relative depression of

tetanic [Ca2+]i is similar at all stimulation frequencies

(Westerblad et al. 1993). Additionally, in single type II

mouse fibres, the EC-induced reduction in maximal

force is only partly restored by adding caffeine,

suggesting a reduction in SR luminal releasable Ca2+

(Ingalls et al. 1998). The reduction in maximal caffeine-

induce force and the uniform reduction in [Ca2+]iassociated with LFF, suggest that LFF is caused by

failure in the excitation–contraction (E–C) coupling

prior to the SR Ca2+ release, e.g. reduced SR content of

releasable Ca2+. However, little is known about the SR

Ca2+ stores during LFF.

In addition to reduced [Ca2+]i, events downstream the

E–C coupling e.g. myofibrillar Ca2+ sensitivity may

cause fatigue. The myofibrillar Ca2+ sensitivity is

reduced by increased Pi and H+ as shown in skinned

fibres (Palmer & Kentish 1994). In line with this, high

intensive isometric stimulations, known to increase Pi

and H+, reduce Ca2+ sensitivity in intact fibres (Moop-

anar & Allen 2006) and studies in intact fibres suggest

that myofibrillar Ca2+ sensitivity also is reduced after

severe EC (Balnave & Allen 1995). However, the effect

of EC on myofibrillar Ca2+ sensitivity has not been

investigated in skinned fibres where the [Ca2+] and

intracellular milieu can be controlled.

The purpose of the present study was to evaluate the

effects of fatiguing EC on Ca2+ handling properties in

mammalian slow twitch muscles. We hypothesize that

EC reduces both the SR content of releasable Ca2+ and

myofibrillar Ca2+ sensitivity.

Experimental design and methods

Experimental design

Animal and muscle handling. Male Sprague-Dawley

rats (n = 24, weighed 215 � 5 g (mean � SEM))

housed and purchased from the Institute of Biomedi-

cine, Odense University Hospital were used. Animals

were housed in cages with a 12 : 12 h light : dark cycle

and provided unrestricted access to water and food. The

animals were delivered to the laboratory at least 3 h

prior to killing by cervical dislocation. Cervical dislo-

cation was conducted in accordance with the guidelines

of the animal ethics committee at Odense University

hospital. Both of the soleus (SOL) muscles were quickly

excised and mounted vertically between a hook and a

force transducer [K30 type351; Hugo Sachs Electro-

nick, March-Hugstetten, Germany, calibrated between

0 and 590 mN (r2 = 0.99)] in a temperature controlled

experimental chamber at 30 �C (Schuler organbad;

Hugo Sachs Electronick), containing Krebs-Ringer buf-

fer continually gassed with a mixture of 95% O2 and

218� 2007 The Authors

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Reduced SR Ca2+ content following eccentric contractions Æ J S Nielsen et al. Acta Physiol 2007, 191, 217–228

5% CO2. The Krebs-Ringer bicarbonate buffer con-

tained (in mm) 120.1 NaCl, 25 NaCHO3, 4.7 KCl, 1.2

KH2PO4, 1.2 MgSO4, 1.3 CaCl2 and 5.0 d-Glucose at

pH 7.3. Muscle contractions were evoked electrically

using 1 ms square pulses at 20 V/cm through two

platinum electrodes located close to either side of the

muscle. Force was recorded on a chart recorder (Kipp &

Zonen BD112, Delft, The Netherlands). The muscle

was gently adjusted to the length at which maximal

twitch force could be recorded, this length is referred to

as the optimal muscle length. After adjusting optimal

length, a control tetanus was evoked (60 Hz, 1.5 s) and

the muscle was allowed to equilibrate for 30 min after

which an additional control tetanus was conducted. The

force during the control tetanus was within 5% of the

initial tetanic force for all muscles.

Eccentric contraction model and stimulation protocol.

After the 30 min equilibration and a force–frequency

protocol (see below), the muscles were attached to a

level transducer (B40 type 373; Hugo Sachs Electro-

nick). The level transducer was placed in a locked

position with muscle length corresponding to 75% of

optimal length (resting position). The level arm was

adjusted by visual inspection to have a range of motion

corresponding to �25% of optimal length. The 30 min

EC protocol consisted of 30 bouts of 20 Hz stimulation

for 2 s once every min. During the first brief period

(�200 ms) of each 2 s stimulation bout, the muscles

contracted isometrically in the resting position. There-

after, a catch was manually released, applying an

external force to the muscle (590 mN, 4.7 cm from

the centre of rotation), thereby stretching it until it was

mechanically blocked at 125% of optimal length. This

EC period lasted between 200 and 1000 ms, depending

on the degree of fatigue. During the remaining part of

the stimulation (1000–1800 ms), the muscle was con-

tracting isometrically at 125% of optimal length.

Immediately after each stimulation bout, muscles were

quickly placed in the resting position. Instantly after the

EC protocol, the muscle was gently transferred from the

level transducer to the isometric transducer, care was

taken not to stretch the muscle during transferring.

Force–frequency protocol in whole muscle. The effects

of EC on muscle contractility in whole SOL muscles

were evaluated using a force–frequency (F–F) protocol

pre-EC and either 0, 1, 2, 3, or 5 h post-EC. The F–F

protocol consisted of five separate 1.5 s stimulation

periods of increasing stimulation frequency (10, 16, 20,

40 and 100 Hz). To avoid that fatigue interfered with

the F–F protocol, a rest period of 30 s was given

between 10 and 16 Hz stimulation periods, and a 60 s

rest period between all others. A 60 s rest period was

sufficient to maintain maximal force during seven

repeated stimulations at 100 Hz in a fresh muscle. As

EC may evoke changes in the serial- and parallel-elastic

parts of the muscle and changes in the mounting that is

unrelated to contractile changes, the optimal muscle

length was adjusted before each F–F stimulation proto-

col (MacIntosh & MacNaughton 2005).

Muscle analysis. Muscles were removed from the

stimulation chamber exactly 2 min after the F–F pro-

tocol either pre-, 0, 1, 2, 3, or 5 h post-EC muscles,

blotted on Whatman paper grade 1 (Whatman Ltd,

Maidstone, UK), placed on a glass plate cooled to 0 �C,

and divided in two specimens by dissecting in the

longitudinal direction. The specimens were randomized

and either (1) frozen for later analyses of metabolites,

(2) homogenized and frozen for later analysis of SR

vesicle Ca2+ uptake and release, or (3) immerged in

paraffin oil (�22 �C) and cooled on ice (5 min) before

immediate determination of SR Ca2+ handling in

mechanically skinned single fibres.

SR vesicle Ca2+ uptake and release measured in crude

muscle homogenate. The technique for measuring SR

vesicle Ca2+ uptake and release has been described

previously in detail (Ørtenblad et al. 2000, Nielsen

et al. 2005). Briefly, the SR vesicle, oxalate mediated,

Ca2+ uptake and release was measured fluorometrically

(Ratiomaster RCM; Photon Technology International,

Brunswick, NJ, USA) at 37 �C using a fluorescent Ca2+

indicator (indo-1). All raw-data of Ca2+ release and

uptake were mathematically fitted using mono expo-

nential equations as previously described (Nielsen et al.

2005). The values obtained for SR Ca2+ uptake and

-release rates are in arbitrary units Ca2+, however,

expressed as lmol Ca2+ mg)1 protein.min)1.

Skinning and mounting of single fibres. Single fibres

were isolated from the muscle and mechanically skinned

under oil. This was achieved by rolling back the

sarcolemma using jewellers forceps as previously des-

cribed (Fink et al. 1986, Lamb & Stephenson 1994).

Due to possible heterogeneity between fibres, owing to

differences in oxygen availability and/or degree of

stimulation, fibres were isolated from both the core

and the outer layer of the muscles. Photographs of each

fibre were taken under oil, using a digital camera

(3· optical magnification; Canon powershot A80,

Tokyo, Japan) through the microscope (50· optical

magnifications), for later determination of fibre cross-

sectional area (CSA). The fibre was then mounted

between a small hook and a force transducer (SensoNor

801, Horten, Norway), calibrated between 0 and

0.78 mN (r2 = 0.99), using fine silk thread, and

stretched to 120% of slack length under oil. All force

recordings were continuously sampled at 1 KHz using

� 2007 The AuthorsJournal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x 219

Acta Physiol 2007, 191, 217–228 J S Nielsen et al. Æ Reduced SR Ca2+ content following eccentric contractions

a custom-made labview program (labview 8.0;

National Instruments, Austin, TX, USA). Between three

and six fibres were skinned from each muscle specimen.

These fibres were analysed consecutively during a

period of 2–4 h. The fibre properties were not changed

by storage in oil for 4–5 h. All single fibre experiments

were performed at room temperature (20–22 �C.).

Solutions for the mechanically skinned fibre prepar-

ation. Solutions for the mechanically skinned fibre

preparation were prepared as described by Stephenson

& Williams (1981). The standard HDTA solutions

contained (mm); K+ (126), Na+ (36), total ATP (8), CrP

(10), Hexamethylene diamine tetraacetate (HDTA2),

50, Fluka, Buchs Switzerland), HEPES (90), total Mg2+

(8.5, giving a free [Mg2+] of 1 mm). Solutions similar to

the HDTA solutions were made in which the 50 mm

HDTA was replaced with either 50 mm EGTA for

strong Ca2+ buffering (EGTA solution) or 50 mm

EGTA plus 48.5 mm added Ca2+ (CaEGTA solution,

pCa 4.7), with total magnesium of 10.3 and 8.1 mm,

respectively, to maintain the free [Mg2+] at 1 mm. pCa

was set by adding appropriate mixtures of the EGTA

and CaEGTA solutions. All solutions contained 1 mm

total EGTA. The osmolality of all solutions was

295 � 5 mOsm kg)1, determined by vapour pressure

osmometry (Wescor 5500, Logan, UT, USA), and pH

was 7.10 � 0.01. The free [Ca2+] in the solutions were

determined using a Ca2+-sensitive electrode (Orion

Research, Boston, MA, USA). pCa in the HDTA

solution with a total amount of 50 lm EGTA was

measured to 6.6, after determining the electrode output

in a similar HDTA solution with 10% total of

CaEGTA/EGTA mixtures set at various levels by

adding 4 : 1, 2 : 1, 1 : 1 mixtures, to give pCa 6.1,

6.4 and 6.7 at pH 7.10. The release solution was

similar to the HDTA solution, but only contained

2 mm Mg2+ (0.05 mm free) and 30 mm caffeine was

added.

Measuring the SR content of releasable Ca2+ in skinned

single fibres. The force–time integral (area) of the

caffeine-induced force response is related to the amount

of releasable Ca2+ in the SR (Endo & Iino 1980,

Launikonis & Stephenson 1997, Trinh & Lamb 2006).

The relative releasable SR Ca2+ content was determined

by measuring the force–time integral of the caffeine-

induced force response, and expressing it either in per

cent of that after maximal SR Ca2+ loading of the same

fibre or force–time integral per fibre CSA. Maximal SR

Ca2+ loading equalled the force–time integral of the

caffeine-induced force response after 240 s loading in

pCa 7.0 (pCa = )log[Ca2+], see Fig. 6).

Comparing the relative force–time integrals presumes

a linear relation between the relative force–time integral

and loading time, which was confirmed between 15 s

and 120 s (Fig. 6). For longer loading times SR was

saturated and the force–time integral is defined as

maximum (Fig. 6). Thus, care was taken that all data

were obtained with SR Ca2+ load within the linear

range. The deviation from linearity at low SR Ca2+

levels could be speculated to be due to back inhibition

of the SR Ca2+-ATPase at eSRCa2+ above 30–60% of

maximum load, thus leading to relatively higher loading

rates at very low eSRCa2+.

The [EGTA] in all solutions was 1 mm, which

ensured a constant Ca2+ buffering and a submaximal

force production during exposure to caffeine. Thus,

preceding the protocol measuring releasable Ca2+ in the

SR, fibres were equilibrated with EGTA for 15 s in the

wash solution (pCa 8.0). To verify that endogenous SR

Ca2+ level was unaffected by the wash solution (i.e.

standard HDTA solution with fixed pCa), resting fibres

were washed in standard HDTA solutions with different

pCa’s after high (64% of max Ca2+ load) or low (33%

of max Ca2+ load) Ca2+ loading (Fig. 1). After loading,

the fibres were immersed in standard HDTA solutions

with pCa of 9.0, 8.5, 8.0, or 7.5 for 15, 30, 60, or 120 s

respectively, before being exposed to the Ca2+ release

solution (Fig. 1). From these experiments a pCa level of

8.0 was chosen for the wash solution.

The endogenous SR content of releasable Ca2+ (eSR-

Ca2+) of single fibres. The fibres mounted between the

hook and the force transducer, were moved from the

paraffin oil into the wash solution (standard HDTA

solution with pCa of 8.0) for 15 s. Whereafter,

release of endogenous SR Ca2+ was initiated by

exposing the fibre to a release solution, containing

30 mm caffeine, for 60 s (Fig. 2). Subsequently, the

fibres were maximal loaded in the load solution

(standard HDTA solution with pCa of 7.0) for 240 s,

before the cycle was repeated by 15 s washing and

60 s exposure to the release solution (see Fig. 2;

Endogenous and 240 s load). The relative SR content

of releasable Ca2+ (eSRCa2+) was then calculated as

described above.

Rate of SR Ca2+ loading in single fibres (SR Ca2+

loading). Following the determination of eSRCa2+, the

fibre was loaded (pCa 7.0) for 15, 30 or 60 s

respectively. Following loading, the fibre was washed

(15 s) and immersed in the Ca2+ release solution

(60 s, Fig. 2). The force–time integral was determined

for each fibre and normalized to both the maximal

SR Ca2+ content (240 s load) and CSA. The mean

relative SR content of releasable Ca2+ of all fibres

loaded at 15, 30, or 60 s was plotted against loading

time and the rate of SR Ca2+ loading calculated by

linear regression. The 30 s loading was performed

220� 2007 The Authors

Journal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x

Reduced SR Ca2+ content following eccentric contractions Æ J S Nielsen et al. Acta Physiol 2007, 191, 217–228

twice (Fig. 2) and used as a quality indicator of the

fibre. If the difference in the force–time integral

between the first and the second 30 s load was

greater than 10%, the fibre was excluded (five fibres).

The coefficient of variation between duplicate deter-

minations of force–time integral after 30 s loading

was 8%, when conducted as shown in Fig. 2.

Myofibrillar Ca2+ sensitivity in single fibres. Following

the measurements of eSRCa2+ and SR Ca2+ loading,

the force–pCa relationship was obtained by measuring

the force in fibres subjected to an series of increasing

[Ca2+] i.e. six solutions from pCa 6.8 to 4.7 prepared

as described by (Fink et al. 1986). The solutions were

prepared in four parts of the standard HDTA solution

and one part of appropriate amounts of CaEGTA and

EGTA solutions, i.e. clamping pCa. Additionally, a

solution without Ca2+ (zero-Ca2+) was prepared

similar to the standard HDTA, but with an [EGTA]

and [Ca2+] of 50 and 0 mm respectively. Prior to

subjecting fibres to the series of increasing pCa, fibres

were immersed twice in pCa 4.7 (CaEGTA solution)

for at least 30 s, separated by a 60 s soaking in the

zero-Ca2+ solution. Fibres were subjected to the series

of increasing pCa twice (separated by 30 s soaking in

zero-Ca2+ solution) and the second used in the

following calculations. Force at the different pCa’s

was expressed relative to the maximal Ca2+ activated

force (pCa 4.7), and fitted to a non-linear sigmoi-

dal dose–response equation with a variable slope

(graphad prism version 4.0; GraphPad Software, San

Diego, CA, USA). From this equation, the Hill

coefficient and the calcium concentration evoking

50% of the maximal Ca2+ activated force (pCa50)

was determined for each individual fibre.

Figure 1 Effect of pCa in wash solution on SR Ca2+ content.

The SR content of releasable Ca2+ was loaded to either

33 � 8% (a, nfibres = 9, nmuscles = 4) or 64 � 11%

(b, nfibres = 11, nmuscle = 5) of the maximal loading capacity

(240 s load) in pCa 7.0. After loading, fibres were

incubated in pCa of 9.0, 8.5, 8.0, or 7.5 for 15, 30, 60, or 120 s

and Ca2+ content of SR determined by releasing Ca2+ (caffeine

and low Mg2+). *Significantly different from other pCa at same

incubation time, #Significant different from 15 s incubation in

the same pCa.

Figure 2 Original trace showing the caffeine-induced force responses from a single fibre. The first peak on the left shows the

initial force response after exposing the fibre to low Mg2+ and high caffeine (Caff). The following force responses were initiated

in the same way after loading (L) for 240, 60, 30, or 15 s in pCa 7.0. Prior to each force measurement the fibre was washed

for 15 s in pCa 8.0 (W) to remove remaining Ca2+ from the incubation solution. The maximal Ca2+ activated force was

measured in pCa 4.7. The initial force, and the force after 240 s loading corresponded to 20% and 50%, of the maximal

Ca2+ activated force respectively. The baseline equals 0 force and stayed constant throughout all experiments.

� 2007 The AuthorsJournal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x 221

Acta Physiol 2007, 191, 217–228 J S Nielsen et al. Æ Reduced SR Ca2+ content following eccentric contractions

Single fibre diameter, cross-sectional area and classifi-

cation. The photograph of each fibre taken under oil

was printed and the fibre diameter was measured at

three locations i.e. the middle, and 34 of the distance

between middle and each end of the fibre. These

measurements were averaged and, assuming that each

fibre was a cylinder, the cross-sectional area (CSA)

was calculated. The maximal Ca2+ activated force (pCa

4.7), from the last of the two measured force–pCa

relation, was expressed per cross-sectional area for each

fibre.

After measuring the force–pCa relation, all fibres

were frozen for later analyses of the myosin heavy chain

(MHC) composition by SDS–PAGE (Betto et al. 1986).

Fibres were run on an 8% polyacrylamide gel for 42 h

as described by (Andersen & Aagaard 2000). Subse-

quently, gels were silver stained using a commercial kit

(Amersham Bioscienes AB, Uppsala, Sweden). Fibres

showing the slightest trace of MHC II (n = 10; hybrid

fibres) or pure MHC II (n = 0) fibres were excluded

from the data set. The SOL fibres analysed (n = 66),

contained 15% hybrid fibres but no pure type II.

Effects of [CaEGTA] on fibre loading. To ensure that

the loading rate was not dependent on the rate of

diffusion of [CaEGTA] into the fibre, the load rate was

measured during conditions of different [CaEGTA] but

constant pCa. In load solutions with EGTA/CaEGTA of

1/0.33, 1.5/0.50 and 2.4/0.80 (i.e. constant PCa 7.0), ST

fibres loaded 64 � 6%, 69 � 4%, 69 � 4% of maxi-

mum load within 30 s loading time respectively (n = 5).

There were no differences in the loading ability between

different [CaEGTA].

Muscle metabolites. Freeze-dried specimens were

removed of all connective tissue and blood and extrac-

ted with HClO4 (0.5 m). The neutralized muscle extract

(KHCO3 2.2 m) was analysed with enzymatic fluoro-

metric methods for ATP, creatine-phosphate (CrP) and

lactate as previously described (Harris et al. 1974).

Statistical analyses. All data are shown as mean �standard error of the mean (SEM). Data were analysed

using unpaired t-test and anova when appropriate. A

Fisher’s PLSD post hoc test was used to find significance

following anova. All analyses were conducted in

statview 5.0 (SAS Institute, Cary, NC, USA). The

significant level was set to P £ 0.05.

Results

Force production in whole muscle

In whole muscles, the EC protocol reduced the maximal

force at 100 Hz by 30% from 550 � 10 mN to

376 � 24 mN (P < 0.0001, Fig. 3a). Maximal force

remained depressed after 3 h but was fully restored

following 5 h recovery. EC reduced force at 10 and

16 Hz force by 52 and 51% respectively, and the

16/100 Hz force ratio by 33% (Fig. 3b). The 16/100 Hz

force ratio was further reduced after 2 h recovery (65%

reduction vs. 0 h post-EC). The 16/100 Hz force ratio

was partially restored after 3 and 5 h recovery, but

remained depressed compared to pre-EC. There were no

changes in maximal force or 16/100 Hz force ratio in

resting muscles incubated for 4 h. These results

demonstrate that the EC protocol caused a pronounced

long-lasting LFF.

(a)

(b)

Figure 3 The maximal force (100 Hz, a) and the 16/100 Hz

force ratio (b) in whole muscles. Force was measured before

and after EC. Both the maximal force at 100 Hz (a) and the

16/100 Hz force ratio (b) is given in percent of the pre EC

values. *Significantly different from pre-EC, �Significantly

different from 0 h post-EC, §Significantly different from 1

and 2 h post-EC. Pre-EC and 0 h Post-EC nmuscle = 26 and

nrat = 15, 1 h Post-EC nmuscle = 8 and nrat = 5, and 2, 3 and

5 h Post-EC nmuscle = 2 (the number of muscles is given in the

figure).

222� 2007 The Authors

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Reduced SR Ca2+ content following eccentric contractions Æ J S Nielsen et al. Acta Physiol 2007, 191, 217–228

Muscle metabolites

Following EC, muscle ATP was reduced from

18.6 � 0.6 to 14.8 � 1.2 mmol kg)1 dw and CrP was

reduced from 40.8 � 2.5 to 29.4 � 3.2 mmol kg)1.

Following EC, lactate increased from 8.1 � 3.0 to

23.5 � 2.5 mmol kg)1 dw. All muscle metabolites were

restored and not different from that at rest after 1 and

5 h recovery.

Endogenous SR Ca2+ content and myofibrillar Ca2+

sensitivity

Prior to stimulation, the relative endogenous SR content

of releasable Ca2+ (eSRCa2+) in mechanically skinned

fibres was 45 � 5% of the maximal SR Ca2+ content

(240 s load). After EC, the eSRCa2+ per fibre CSA was

reduced by 38% (P = 0.05, Fig. 4). The maximal Ca2+

loading capacity of SR was reduced by 32% following

EC (Fig. 6b). Hence, EC clearly decreases eSRCa2+ both

when expressed per fibre CSA and relative to maximum

loading ability pre-EC, and further the maximum

loading ability is depressed by EC.

The force–pCa relation was determined by exposing

single fibres to increasing [Ca2+] (pCa from 6.8 to 4.7).

The Hill coefficient and the [Ca2+] eliciting 50% of the

maximal Ca2+ activated force (pCa50) were calculated

for each individual fibre. Both the Hill coefficient (pre:

4.23 � 0.30 and post: 4.66 � 0.49) and the pCa50 (pre:

6.10 � 0.03 and post: 6.12 � 0.03) were unaffected by

EC (Fig. 5). The maximal Ca2+ activated force (in pCa

4.7) was 0.34 � 0.03 and 0.28 � 0.03 mN pre- and

post-EC respectively. The average fibre radius was

30.0 � 2.4 and 28.3 � 1.2 lm pre- and post-EC

respectively, and not affected by EC (pre- vs. post-

EC). The maximal force normalized to fibre cross-

sectional area was 121 � 13 and 113 � 14 mN mm)2

pre- and post-EC respectively. The maximal Ca2+

activated force measured both per fibre or per cross-

sectional area was not significantly different when

comparing pre- and post-EC values.

SR Ca2+ pump function and SR vesicle release in

homogenate

The SR Ca2+ pump function was measured in crude

muscle homogenate as the rate of SR vesicle Ca2+

uptake (Table 1) and was unaltered by EC. The SR

Ca2+ pump function was also measured as the SR Ca2+

loading ability in mechanically skinned single fibres.

The fibres were loaded in pCa 7.0 for 15, 30, or 60 s

and the relative content of releasable Ca2+ in SR

measured (Fig. 6a). The SR Ca2+ loading ability of the

fibres was unaffected by EC, i.e. no significant differ-

ences between the regression lines or individual time

points when comparing data pre- and post-EC. When

expressing the loading ability per CSA, there were no

change in eSRCa2+ at 15, 30 and 60 s loading, however,

the maximum loading ability per fibre CSA was 32%

lower following EC (Fig. 6b). There was no significant

difference in maximal rate of SR vesicle Ca2+

release in crude muscle homogenate at nadir [Ca2+]

(Table 1).

Figure 4 The endogenous SR content of releasable Ca2+. A

plot of the endogenous SR content of releasable Ca2+ (eSR-

Ca2+) measured in mechanically skinned fibres pre- and post-

EC. The line represents the mean, each bar is the SEM, and

each dot represents the eSRCa2+ of a fibre. Pre-EC (nfibres = 21,

nmuscles = 12, nrats = 12) and post-EC (nfibres = 26,

nmuscles = 10, nrats = 10). All fibres were classified as MHC I by

SDS–PAGE. *Significantly different from pre-EC (P = 0.05)

using unpaired t-test.

Figure 5 Myofibrillar Ca2+ sensitivity in mechanically skinned

fibres. The force–pCa relation in mechanically skinned fibres

measured pre- and post-EC. There was no significant effect of

EC on the force–pCa relation, i.e. the Hill coefficient, or the

pCa50. Furthermore, the maximal Ca2+ activated force per

cross section area were unchanged from pre- to post-EC.

Pre-EC (nfibres = 19, nmuscles = 9, nrats = 8) and post-EC

(nfibres = 17, nmuscles = 11, nrats = 11).

� 2007 The AuthorsJournal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x 223

Acta Physiol 2007, 191, 217–228 J S Nielsen et al. Æ Reduced SR Ca2+ content following eccentric contractions

Discussion

The major finding was that EC reduced the SR content

of releasable Ca2+, but that neither the Ca2+ sensitivity

of the contractile proteins nor the maximal Ca2+

activated force were affected by EC.

Endogenous SR content of releasable Ca2+ and

myofibrillar Ca2+ sensitivity

Stimulation of whole muscle to fatigue resulted in a

pronounced decrease ()38%) in the eSRCa2+. This is, to

the best of our knowledge, the first study to demonstrate

reduced eSRCa2+ at fatigue in mammalian muscle

fibres. Studies in single fibres from cane toad have

shown that caffeine-induced SR Ca2+ release (i.e.

eSRCa2+) decreased by 46% after fatiguing isometric

stimulation (Kabbara & Allen 1999). This was further

corroborated by the finding that the SR [Ca2+] depend-

ent fluorescent signal measured in intact fibres,

decreased with 29% after fatiguing isometric contrac-

tion (Kabbara & Allen 2001). We have in the present

study examined eccentric–isometric contraction in slow-

twitch fibres from soleus muscle. The basis for this

protocol is that we wanted to maximize LFF, since this

phenomenon relates to disturbances in Ca2+ handling

(Ingalls et al. 1998) and is more accentuated after EC

than after concentric contraction (CON) (Jones et al.

1989). The protocol was also designed to minimize

metabolic perturbations due to its impact on muscle

contraction. Metabolic perturbations are less prominent

after EC (vs. CON) and in slow-twitch fibres (vs. fast-

twitch fibres). Further studies are required to investigate

if reduced eSRCa2+ also occurs after concentric con-

tractions and in fast-twitch muscles and thus is common

feature of fatigue.

Maintained Ca2+-sensitivity is a prerequisite for

determination of eSRCa2+ with the technique used in

this study. Ca2+ sensitivity was determined in mechan-

ical skinned fibres exposed to different [Ca2+]. Neither

Table 1 SR vesicle Ca2+ uptake and release

Pre-EC Post-EC

Rate of Ca2+ uptake at 800 nm Ca2+ (lmol mg)1 prot. min)1) 5.4 � 0.2 5.4 � 0.5

Rate of Ca2+ uptake at 200 nm Ca2+ (lmol mg)1 prot. min)1) 1.3 � 0.1 1.3 � 0.1

s, the inverse rate constant (s) 23.9 � 1.9 25.2 � 2.8

Rate of Ca2+ release at nadir Ca2+ (lmol mg)1 prot. min)1) 3.5 � 0.1 3.8 � 0.3

Rate of Ca2+ release at 200 nm (lmol mg)1 prot. min)1) 2.7 � 0.1 3.0 � 0.2

Nadir[Ca2+] (nm) 3.8 � 0.6 5.5 � 2.0

The SR vesicle Ca2+ uptake and release were analysed fluorometrically, in crude muscle homogenate. The s (tau) is the inverse rate

constant representing the time for 63% of the Ca2+ to be taken up by the SR vesicles. The nadir[Ca2+] is the [Ca2+] before initiating

Ca2+ release. The low values indicate that the vesicles took up all Ca2+ and that the experimental condition during the assay was the

same pre- and post-EC.

Figure 6 SR content of releasable Ca2+ in resting skinned

single fibres and after EC. The force–time integral (area) of the

caffeine-induced force response in mechanically skinned fibres

loaded in pCa 7.0 for 15, 30, 60, 120, or 240 s. After the

loading fibres were briefly washed (pCa 8.0 for 15 s), before

Ca2+ was released in high caffeine and low Mg2+ (see Fig. 2).

The force–time integral was expressed both relative to the

force–time integral after maximal Ca2+ loading of the same

fibre, i.e.240 s loading (a) and as force–time integral per fibre

CSA (b). In one fibre (pre-EC), the SR was loaded for 120 ((x))

which corresponded to 87% of the maximal Ca2+ load. The

variation in maximal SR Ca2+ content (240 s load) represents

the over all variation among fibres. *Significantly different

from pre-EC. Figure a and b rest, nfibres = 19, nmuscles = 10,

nrats = 9; Figure a and b post-EC, nfibres = 20, nmuscles = 8,

nrats = 8.

224� 2007 The Authors

Journal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x

Reduced SR Ca2+ content following eccentric contractions Æ J S Nielsen et al. Acta Physiol 2007, 191, 217–228

Hill coefficient, pCa50 nor the maximal Ca2+ activated

force were affected by EC, demonstrating that myofi-

brillar Ca2+ sensitivity was unchanged. This is consis-

tent with previous findings in intact fibres after

isometric contractions (Westerblad et al. 1993), and

of stimulated over-stretched fibres (EC model)

(Balnave & Allen 1995). Both of these studies

demonstrated LFF. Taken together, these studies dem-

onstrate that the observed reduction in force during

LFF is due to other factors than reduced myofibrillar

Ca2+ sensitivity.

When eSRCa2+ is reduced experimentally the SR

Ca2+ release during a twitch is attenuated (Posterino &

Lamb 2003). Studies in single fibres (Westerblad et al.

1993) have shown that the decline in force is related to

reduced tetanic [Ca2+]i, and the observed reduction in

eSRCa2+ can therefore be an important cause of

fatigue. The tetanic [Ca2+]i is reduced to a similar

extent at all stimulation frequencies in fatigued single

fibres (Westerblad et al. 1993). This will, due to the

sigmoidal shape of the force–pCa relation, explain the

more pronounced decline in force at low frequencies

(i.e. LFF).

Possible explanations for reduced eSRCa2+

The observed reduction in eSRCa2+ may be explained

by precipitation of Ca2+-P in SR. The Ca2+-P precipi-

tation theory (Fryer et al. 1995) has been widely

accepted and used to explain the reduced SR Ca2+

release in fatigued fibres [review see (Steele & Duke

2003,Allen et al. 2002)]. There is some experimental

support for the hypothesis, in that experimental increa-

ses of Pi have been shown to reduce tetanic [Ca2+]i in

both intact (Westerblad & Allen 1996b) and skinned

fibres (Fryer et al. 1995). From our data, it can be

calculated that the summed decrease in ATP and CrP,

assuming degradation of ATP to inosine monophos-

phate, corresponds to an increase of Pi of 19 mmol kg)1

dry muscle or �6 mm assuming 3 L of water kg)1 dry

muscle. This is much less than that observed after

anaerobic isometric contraction (Sahlin et al. 1987) or

after concentric contractions (Beltman et al. 2004). It

may be argued that the decline in PCr and thus the

increase in Pi is much larger immediately after EC and

that PCr is restored during the 6-min period elapsed

before muscle samples were frozen. However, studies

on mechanically skinned fibres show that the caffeine-

induced SR Ca2+ release recover after Pi exposition

within 7 min (Posterino & Fryer 1998). Recovery in

PCr would therefore also be paralleled by removal of

Ca2+-P precipitation. Although we cannot exclude that

Ca2+-P precipitation occurs with the present EC proto-

col, the relatively small increase in Pi compared with

other types of exercise/stimulation protocols suggest

that other mechanisms contribute to the observed

reduction in eSRCa2+.

Skeletal muscle mitochondria may, besides their

important role as energy suppliers, play a role in

modulating [Ca2+]i (Sembrowich et al. 1985, Bruton

et al. 2003). Several groups have reported that mito-

chondria isolated from skeletal muscle contain more

Ca2+ after exhaustive exercise than control muscle

(Duan et al. 1990, Madsen et al. 1996). Thus, Ca2+

sequestration by the mitochondria during EC could at

least in part explain the observed decrease in eSRCa2+,

i.e. due to loss of Ca2+ from the SR.

Modification of the main intra-SR Ca2+ buffer calse-

questrin (CSQ) could be involved in the reduced

maximal SR loading of Ca2+ after EC. The present

results may add-on to the evolving concept of CSQ as a

dynamic store, observed in both vesicular SR and

skinned skeletal muscle fibres (Ikemoto et al. 1991,Lau-

nikonis et al. 2006). According to the concept, CSQ in

the terminal cisternae of the SR, forms linear polymers

which is favoured by Ca2+ binding. During Ca2+ release,

[Ca2+]SR will decrease, causing widespread depolymer-

ization of CSQ with loss of binding sites and thus a

decreased SR Ca2+ buffering capacity (Park et al. 2003,

Launikonis et al. 2006). Continuous SR Ca2+ release, as

with tetanic contractions, causing rapid decay in

[Ca2+]SR will cause a further CSQ depolymerization,

hence, less stored [Ca2+]SR. However, our work offers

no evidence for a specific nature of this dynamic Ca2+

store buffering by CSQ, which therefore remains

speculative.

A slower SR Ca2+ uptake is a potential explanation

for the reduction in eSRCa2+ at fatigue. A reduced rate

of vesicle SR Ca2+ uptake is often observed after

fatiguing isometric and concentric exercise (Booth et al.

1997, Steele & Duke 2003). After exercise involving

EC there are some studies showing an increased rate of

vesicle SR Ca2+ uptake (Schertzer et al. 2004) whereas

other studies (Ingalls et al. 1998), including the present

study, show no change. The function of SR Ca2+ pump

was, beside in vitro vesicle measurements, in this study

measured also in single fibres. The relative rate of SR

Ca2+ loading ability was unchanged following EC

(Fig. 6a) but the maximal SRCa2+ loading ability was

reduced (Fig. 6b). The latter finding may relate to

increased Ca2+ leakage from the SR e.g. increased SR

pump slippage. SR pump slippage is more likely to

occur at high rather than low Ca2+ gradients between

SR lumen and the load solution, i.e. near maximum

load. An increased Ca2+ leakage would prevent large

gradients and increase nadir [Ca2+] in the load solution

during measurements with SR vesicles. However, our

results from SR vesicles, Ca2+ buffered with oxalate,

could not demonstrate any difference between pre- and

post-exercise in nadir [Ca2+], which would increase

� 2007 The AuthorsJournal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x 225

Acta Physiol 2007, 191, 217–228 J S Nielsen et al. Æ Reduced SR Ca2+ content following eccentric contractions

with an increased leak to pump ratio. The similar SR

Ca2+ loading rates before and after EC in skinned fibre

preparation and in SR vesicles does not exclude that

the SR Ca2+ uptake in vivo is reduced at fatigue. It is

well known that increases in ADP (Macdonald &

Stephenson 2001) and possibly H+, which are associ-

ated with fatigue, may impair pump function with an

ensuing decrease in SRCa2+. Reduced SR Ca2+ pump

function in vivo cannot explain the reduced maximal

loading capacity observed in vitro but may explain the

reduced relative loading. However, the observed chan-

ges in metabolites were rather small and muscle

samples were frozen 6 min post-exercise and therefore

it appears unlikely that this is the cause of reduced

eSRCa2+.

Other potential sites of fatigue

In addition to the eSRCa2+ and myofibrillar Ca2+

sensitivity discussed above, the observed reduction in

force following EC may involve other steps in E–C

coupling. In this study, steps downstream to eSRCa2+ in

the E–C coupling i.e. rate of SR vesicle Ca2+ release, SR

leak and SR pump function were measured. Alteration

in the rate of SR vesicle Ca2+ release measured in crude

muscle homogenate, is thought to be related to intrinsic

changes in the SR Ca2+ release channels (Williams &

Klug 1995). In this study, we found no change in the

rate of SR Ca2+ release measured in vitro in SR vesicles.

This is consistent with other studies using type II

muscles from mice (Ingalls et al. 1998) and human

studies where biopsies were taken pre- and post-EC in

vastus lateralis (Nielsen et al. 2005). Thus, intrinsic

changes to the SR Ca2+ release channels is unlikely to be

the cause of reduced tetanic [Ca2+]i and force. However,

the results from these in vitro methods cannot exclude

that the SR Ca2+ release channels are affected in vivo

but that these alterations are reversed during the

preparation of SR vesicles.

Fatigue may also relate to impaired E–C coupling

upstream of eSRCa2+ including disruption of signal

transmission from t-tubuli by high mechanical load and/

or activation of Ca2+-induced proteases (Murphy et al.

2006). Signs of muscle damage are frequently observed

after intensive contractions, especially when the exercise

includes eccentric components and when fast-twitch

fibres are investigated (Friden & Lieber 2001). Muscle

damage is associated with increased muscle Ca2+ (Gissel

& Clausen 2000), which is the opposite of the present

findings. This apparent contradiction can be explained

by the less severe exercise used in the present study,

where the depressed contractile function was reversible.

We have investigated muscle morphology with trans-

mission electron microscopy and preliminary data from

a few muscles pre- and post-EC show no disturbances in

the structural components or signs of muscle damage

after EC (data not shown).

Endogenous SR Ca2+ content at rest

The eSRCa2+ in non-stimulated type I SOL fibres was

45% of the maximal loading capacity. This is similar to

that observed previously in skinned SOL type I fibres,

where Ca2+ was released with Triton (Fryer & Stephen-

son 1996). Recently, eSRCa2+ was estimated in fibres

from different non-stimulated rat muscles with a similar

technique as used in the present study (Trinh & Lamb

2006). In fast-twitch fibres (type II) from various

muscles [extensor digitorum longus (EDL), proneus

longus, gastrocnemius] eSRCa2+ was only 22% of the

maximal loading capacity, whereas type I fibres from

SOL was 87% loaded. Preliminary results from our

laboratory give a similar value of eSRCa2+ in type II

fibres from EDL (30%, n = 2) but eSRCa2+ in SOL

presented in this study (45%) is considerably lower than

that of Trinh and Lamb (Trinh & Lamb 2006). The

discrepancy may be ascribed to different ways to

calculate maximal loading capacity. In the study by

Trinh & Lamb (2006) a number of SOL fibres were

unable to reload Ca2+ to the endogenous level and it

was speculated that these fibres were necrotic due to

repeated exposure to high [Ca2+]. Pilot studies in our lab

confirmed that SOL fibres loaded at high [Ca2+]

(PCa = 6.7) were unable to load Ca2+ to the initial

maximal and/or submaximal level. We therefore used a

lower [Ca2+] to load SR, i.e. pCa 7.0 vs. 6.7 (Trinh &

Lamb 2006) and only a few fibres showed an impaired

loading capacity. Fibres unable to reach initial Ca2+

loading level were in the present study excluded (few

fibres), whereas Trinh & Lamb (2006) assumed these

fibres to be 100% loaded. The difference in eSRCa2+

between studies can most likely be ascribed to this

methodological difference.

Conclusion

Endogenous SR content of releasable Ca2+ decreased

markedly after EC, whereas myofibrillar Ca2+ sensitivity

and SR Ca2+ kinetics remained unchanged. We suggest

that the decrease in eSRCa2+ results in reduced Ca2+

release and contributes to long-lasting low frequency

fatigue. Further studies are required to investigate if the

phenomenon occurs after other types of contractions

(concentric) and in fast-twitch fibres and thus is a

common feature of fatigue. The results from this study

demonstrate that the SR may be submaximally loaded

at rest. A submaximally loaded SR could act as

a powerful cytosolic Ca2+ buffer, which could

stabilize cytosolic Ca2+ and protect the muscle from

Ca2+-induced necrosis.

226� 2007 The Authors

Journal compilation � 2007 Scandinavian Physiological Society, doi: 10.1111/j.1748-1716.2007.01732.x

Reduced SR Ca2+ content following eccentric contractions Æ J S Nielsen et al. Acta Physiol 2007, 191, 217–228

Conflict of interest

There are no conflicts of interest.

The authors express there gratitude to the ministry of culture,

the committee on sports research for financial support

(TKIF2005-049). Furthermore, we wish to express our appre-

ciation to Joachim Nielsen for performing the TEM analysis.

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