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: [email protected]
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
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
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
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
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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