Elevation of Mitochondrial Ca²⁺ Above a Plateau Level Impairs Force Production and Accelerates Fatigue in Mouse Soleus Muscle

Abstract

Soleus muscle fibres display modest changes in tetanic force and [Ca²⁺]_i during repeated contractions. In this study, we investigate whether increasing mitochondrial Ca²⁺ load during repeated contractions could induce premature fatigue. Intact, single fibres were dissected from the soleus muscles of adult mice. Mitochondrial Ca²⁺ was measured with rhod-2 in intact fibres. Fatigue was induced by 70 Hz, 350 ms tetani given at 2 s intervals in the absence and presence of 10 µM CGP-37157, a potent inhibitor of the mitochondrial Na⁺-Ca²⁺ exchanger. In soleus fibres fatigued in the absence of CGP-37157, tetanic force was significantly reduced by about 30% at the end of the fatiguing stimulation, while mitochondrial [Ca²⁺] increased to a maximum after about 50 tetani and returned to its resting level within 20 min after the end of the stimulation. In the presence of CGP-37157, the maximal mitochondrial [Ca²⁺] increase was more than twice that in control fibres. In addition, fatigue developed more rapidly and force remained depressed after the end of the stimulation. No difference in mitochondrial membrane potential or ROS production was seen between control and CGP-37157 conditions. We conclude that while modest increases in mitochondrial Ca² may be beneficial, excessive mitochondrial Ca² loading depresses muscle function.

1. Introduction

In mammalian postural muscles, fibres are rich in mitochondria with a higher density of mitochondria found closer to the sarcolemma than deeper in the fibre [1,2]. The soleus is an archetypical slow-twitch muscle where mitochondria contribute 5% to 10% of the total fibre volume, greater than that seen in type IIb fast-twitch, glycolytic muscles such as the EDL (2–3%). It should also be noted that the highest density of mitochondria is found close to the sarcolemma and the transverse tubules. Both type I and type IIa fibres have a similar arrangement of mitochondrial columns arranged across the long axis of the fibre. Type I fibres have additional prominent rows of longitudinally orientated connected mitochondria [3]. Due to the dense packing of the myofibrils and the calcium-packed sarcoplasmic reticulum, mitochondria have little room for movement. Unlike other tissues, where mitochondria have a dynamic ever-changing interconnected tubular network (mediated by members of the dynamin superfamily), mitochondria in mature skeletal muscles form relatively stable networks [4]. In addition to mitochondria clustering near the transverse tubules that conduct the action potential deep into the muscle fibre, close contacts between mitochondria, transverse tubules as well as the sarcoplasmic/endoplasmic reticulum (SR) have been described [5]. Extensive connections between the SR and the outer mitochondrial membrane with the assistance of a chaperone protein have been described in heart and other tissues. Separate distinct membrane contacts exist between the transverse tubules and mitochondria. Tunnelling or transfer of Ca²⁺ in rested, non-stimulated mitochondria has been visualised using mitochondrial-targeted indicators [6]. In muscle, mitochondria are perfectly placed to respond to action-potential induced Ca²⁺ release through the ryanodine receptors of the SR by taking up calcium via the uniporter which permits increased mitochondrial enzyme production of ATP [7,8,9]. Several mitochondrial dehydrogenases involved in the tricarboxylic acid cycle are known to require Ca²⁺ [10,11]. Training is known to enhance mitochondrial number and function and thus, to improve ATP generation in muscle and brain [12,13]. During demanding exercise, the muscles’ energy demands exceed the mitochondria’s capacity to produce ATP via oxidative phosphorylation and anaerobic respiration becomes more important. A consequence of this is accumulation of lactate, hydrogen and phosphate ions which together with an increased mitochondrial reactive oxygen species (ROS) production may contribute to the development of muscle fatigue [14,15,16]. However, whether excessive entry of calcium into mitochondria plays a role in muscle fatigue has not been examined previously.

It is well documented that mitochondria take up limited amounts of Ca²⁺ and thus modulate cytosolic-free calcium concentration ([Ca²⁺]_i) in nerves [17], kidneys [18], and muscle [19,20]. A prolonged or uncontrolled increase in mitochondrial Ca²⁺ is considered likely to induce a decline in cellular function and accelerate senescence either directly or by increasing reactive oxygen species (ROS) generation [21,22], which in turn impairs muscle function and damage [23,24,25,26]. Mitochondria isolated from skeletal muscle after exhaustive exercise where force production is impaired contain more Ca²⁺ than those isolated from non-exercised muscle [27,28]. However, even after prolonged periods of stimulation of the nerve or muscle, mitochondrial Ca²⁺ accumulation is limited, reaching a plateau level and thus indicating perfect balance between uptake and extrusion mechanisms [20,29]. It may be speculated that this asymptotic behaviour is a protective mechanism that keeps mitochondrial Ca²⁺ below dangerous levels that would trigger excessive production of ROS or initiate apoptosis. In many tissues including nerve and muscle, extrusion of Ca²⁺ from the mitochondria is predominantly mediated by a Na⁺-Ca²⁺ exchanger that is inhibited by the benzothiazepine compound, CGP-37157 [29,30]. We hypothesise that blocking mitochondrial Na⁺-Ca²⁺ exchange during repeated tetanic contractions would result in excessively elevated levels of mitochondrial Ca2+ and thus accelerate fatigue development. We used fibres from the NMRI mouse soleus muscle because it is composed almost entirely of oxidative type I and oxidative-glycolytic type IIa fibres that are resistant to the development of fatigue due to the relative abundance of mitochondria. With abundant mitochondria to accumulate Ca²⁺, soleus fibres would provide a good model to investigate whether excessive accumulation of mitochondrial Ca²⁺ promotes faster development of fatigue.

2. Materials and Methods

Young (3–5 months) NMRI male mice were supplied by B & K Universal, Sollentuna, Sweden. Twenty mice were used in the experiments reported here. The studies were approved by the Swedish Ethical Review Authority (1) N117/06 Approval Date: 29 March 2006 and (2) N152/11 Approval Date: 13 May 2011. Mice were euthanized by rapid neck disarticulation, and the soleus muscles were removed. The soleus muscle in NMRI mice is composed of type I and type IIa fibres [31]. Single fibres were isolated from the muscles and mounted in a muscle bath as described previously [32]. Fibres were used only if tetanic force was greater than 350 kPa and remained stable (±5%) during the 30 min equilibration period prior to the start of the experiment.

Tetanic contractions were evoked using 70 Hz, 500 ms stimulus trains and fibres were stimulated with up to 700 tetani given at 2 s intervals. Stimulation was stopped when force fell to 40% of the initial force.

2.1. Solutions

Muscle fibres were superfused with a Tyrode solution containing (mM) NaCl 121, KCl 5, MgCl₂ 0.5, Na₂HPO₄ 0.4, CaCl₂ 1.8, EDTA 0.1, NaHCO₃ 24, glucose 5.5 and foetal calf serum (0.2%, Gibco, Billings, MT, USA) and bubbled with 95% O₂/5% CO₂ (pH 7.4). All experiments were performed at room temperature (24–26 °C). A stock solution of 10 mM CGP-37157 (CGP, Tocris Bioscience, Bristol, UK) in DMSO was prepared and 10 µM CGP was applied for 15 min before induction of fatigue. Control fatigue runs were performed in a Tyrode solution containing 0.1% DMSO.

2.2. Measurement of Mitochondrial Ca²⁺, Reactive Oxygen Species, and Mitochondrial Membrane Potential Using Indicators and Laser Confocal Microscopy

To measure mitochondrial Ca²⁺, soleus fibres were incubated in 5 µM rhod-2-AM (Molecular Probes, Leiden, The Netherlands) for 90–120 min at room temperature. Rhod-2 preferentially loads into mitochondria with relatively little left in the myoplasm. It is resistant to photobleaching and gives a large increase in fluorescence upon binding to calcium [32,33]. In a separate series of experiments, soleus fibres were incubated for 10 min at room temperature with 5 µM MitoSox Red (Molecular Probes) which is a specific mitochondrial dye used to monitor mitochondrial reactive oxygen species (ROS). To measure mitochondrial membrane potential, soleus fibres were loaded with 2.6 µM rhodamine 123 (R123; Molecular Probes) for 10 min at room temperature. Following loading with the AM indicators, fibres were washed for at least 30 min. An X-Y (1024 × 1024 pixels) confocal image of the rested fibre was first obtained and fifteen min later, a second confocal image of the rested fibre in either the absence or presence of CGP was obtained and then the fibre was fatigued with a train of repeated tetanic contractions. Stimulation was paused for 6 s and images were taken after 10, 25, 50, 100, 200, 300 and 500 tetani and lastly when the stimulation was ended. During the recovery period after stimulation had ended, X-Y images were taken at regular intervals until the mitochondrial signal returned to its pre-fatigue level.

A BioRad MRC 1024 unit (Biorad Microscopy Division, Hertfordshire, UK) with a krypton/argon mixed-gas laser run at 5 mW and attached to a Nikon Diaphot 200 inverted microscope (Nikon Europe B.V. Amsterdam, The Netherlands) was used. In most experiments, a Nikon Plan Apo 40x oil immersion objective lens (N.A. 1.3, Nikon Europe B.V. Amsterdam, The Netherlands) was used. rhod-2 was excited with 568 nm light and the emitted light collected through a 585 nm long-pass filter. Both MitoSox Red and Rhodamine 123 were excited with 488 nm light and the emitted fluorescence was collected through a 522 nm bandpass filter. The minimum laser power required for acceptable images was 3% of the maximum, although up to 10% power was used. Images were stored and analysed offline with FIJI (NIH, Bethesda, MD, USA, https://hpc.nih.gov/apps/Fiji.html), accessed on 14 April 2026. No noise correction or smoothing was applied to the acquired images. To measure changes in mitochondrial fluorescence, regions of interest were defined in areas containing visible mitochondria close to the sarcolemma and the mean pixel density was calculated. Typically, at least three such regions (1000–2500 µm²) were measured in each fibre. Background fluorescence (the result of intrinsic fluorescence of proteins) was defined as that measured in the myoplasm which had no visible mitochondria. It should be noted that the intrinsic noise of the PMT and the rest of the acquisition system is low and is assumed to be equal in measured regions. Repeated tetanic contractions caused a clear increase in mitochondrial Rhod-2 fluorescence in soleus fibres, while the myoplasm, i.e., background intensity, showed little change. At each time point, the myoplasmic intensity was subtracted from the mitochondrial intensity. Changes in mitochondrial fluorescence intensity at each time point (F) were expressed as a ratio of that measured in the rested fibre (F₀). This procedure allowed comparison of the mitochondrial fluorescence signal recorded in different fibres.

2.3. Statistics

No randomisation regarding fibre allocation to the control group or drug group was done. Experiments were done sequentially, i.e., control, drug, control, etc. All data are expressed as mean ± s.e.m. Student’s t-test (unpaired or paired as necessary) and a repeated-measures ANOVA (when appropriate) were used to check for statistically significant differences (Sigmaplot, 12.5 Systat Software Inc., San Jose, CA, USA). The significance level was set at p < 0.05. As part of the analysis process, a Sigmaplot check was done for the assumptions of the chosen statistic. Data presented here were analysed as follows: For Figures 2, 3A and 4, a one-way repeated measures ANOVA was employed. The Holm–Sidak method was used for post hoc analyses. In Figure 3B, a paired Student’s t-test was used and in Figure 5 an unpaired Student’s t-test was used.

3. Results

Changes in Mitochondrial Ca²⁺ Induced by Repeated Tetani Are Increased by the Presence of CGP-37157

To demonstrate that CGP-37157 could increase mitochondrial Ca²⁺ uptake, soleus fibres loaded with rhod-2 were subjected to two series of 25 tetani, the first in Tyrode only and the second series one hour later in the presence of CGP-37157. Figure 1 shows that there was an increase in the rhod-2 signal, indicating increased mitochondrial Ca²⁺, in the mitochondria close to the sarcolemma in both series of 25 tetani. This increase was more pronounced in the presence of CGP-37157 than in Tyrode. After the series of 25 tetani, the mitochondrial Ca²⁺ signal returned to the resting level more rapidly in the Tyrode solution than in the presence of CGP-37157, a further demonstration of the inhibitory effect of CGP-37157 on Ca²⁺ extrusion from the mitochondria.

Next, soleus fibres loaded with rhod-2 were subjected to a longer series of repeated tetanic contractions to induce muscle fatigue. In control fibres fatigued in Tyrode solution, mitochondrial rhod-2 F/F₀ reached its maximum value of 7.4 ± 3.6 after 50 tetani (Figure 2A). Thereafter, mitochondrial Ca²⁺ declined slightly throughout the rest of the series and at the end of the stimulation, mitochondrial rhod-2 F/F₀ had fallen to 57 ± 1% of its maximum value. At the end of the stimulation, force had decreased to 66 ± 6% of initial tetanic force (Figure 2B). In soleus fibres exposed to CGP-37157, the changes in both mitochondrial Ca²⁺ and tetanic force were more pronounced. After 50 tetani, the mitochondrial rhod-2 signal was already above 10, and it continued to increase until at 300 tetani, mitochondrial rhod-2 F/F₀ had peaked at 18.0 ± 3.2 and tetanic force had declined to 46 ± 5% (Figure 2A). In contrast to the control fibres, none of the six soleus fibres exposed to CGP-37157 were able to maintain tetanic force above the cutoff of 40% (Figure 2B) and the stimulation ended on average after 452 ± 62 tetani. When the stimulation ended, the mitochondrial rhod-2 F/F₀ was 17.0 ± 3.4, 94% of its peak value while tetanic force had decreased to 33 ± 4% of its starting value. A scatterplot of tetanic force versus mitochondrial rhod-2 signal (Figure 2C) showed a general trend for a reduced tetanic force as the rhod-2 signal increased.

In control fibres after the end of the stimulation, mitochondrial rhod-2 F/F₀ returned to its resting value within 20 min (Figure 3A). Tetanic force measured at 10 min post stimulation was 88 ± 8% and recovered completely to its resting value after 30 min (Figure 3B). The situation in CGP-37157 exposed soleus fibres was very different where mitochondrial rhod-2 F/F₀ remained elevated (5.0 ± 1.5) even 30 min after the end of the stimulation and when tetanic force had recovered to only 48 ± 2% of its pre-fatigue value. Thus, the excessive Ca²⁺ loading of the mitochondria that occurred in the presence of CGP-37157 results in a rapid decline of force during fatigue and poor recovery of force afterwards.

The above experiments demonstrate that repeated tetanic stimulation induced Ca²⁺ loading of the mitochondria and this Ca²⁺ load could be markedly increased with CGP-37157 present. Thus, we investigated whether this increased Ca²⁺ load had altered the mitochondrial membrane potential which would impact the mitochondrial ATP production [34]. Using the mitochondrial membrane potential probe rhodamine 123, we found that there was little change in the mitochondrial membrane potential in the soleus fibres during a series of 500 tetani in either the absence or presence of CGP-37157 (Figure 4).

Lastly, we investigated whether the greater mitochondrial Ca²⁺ loading in the presence of CGP-37157 was associated with greater levels of ROS. At the end of the fatiguing protocol, there was a modest non-significant change in ROS in the soleus fibres (Figure 5). Importantly, there was no difference in the ROS signal between the control soleus fibres and those exposed to CGP-37157. Thus, excessive Ca²⁺ loading was not associated with greater levels of ROS production in soleus fibres.

4. Discussion

The major results of this study show that (i) mitochondrial Ca²⁺ increased during periods of stimulation in mouse soleus fibres and this was augmented by CGP-37157 and (ii) this increased mitochondrial Ca²⁺ accumulation was accompanied by a marked impairment of contractile function. The direct stimulation of the muscle sarcolemma with brief electrical pulses allows one to be sure that these effects are happening within the muscle fibre itself and are not due to failure of transmission at the neuromuscular junction.

Removal of Ca²⁺ from the mitochondria is accomplished predominantly by a Na⁺-Ca²⁺ exchanger, which is at least ten times slower than the uniporter-mediated uptake in skeletal muscle [19,35]. Thus, significant mitochondrial Ca²⁺ uptake via the mitochondrial uniporter can occur during repeated electrical stimulation [35], such as used in the present study, due to the high [Ca²⁺]_i (>1 µM) achieved in each tetanus coupled with the slower extrusion of Ca²⁺ from the mitochondria. Some degree of Ca²⁺ uptake by muscle mitochondria during activity appears essential for metabolic function as mitochondrial calcium uniporter knockout mice show a reduced exercise capacity [36]. The decline in mitochondrial [Ca²⁺] that occurred after 100 tetani of the series is not due to reduced [Ca²⁺]_i as the Ca²⁺ transient amplitude shows no decrease during this period [32]. The decline might reflect significant buffering within the mitochondria via Ca²⁺-binding proteins such as calmitine found in the mitochondrial matrix [37,38] or cardiolipin, a negatively charged phospholipid found on the inner mitochondrial membrane [24]. It has been suggested previously that high levels of mitochondrial Ca²⁺ may bind to phosphate and form calcium phosphate compounds [29] and thus limit the free mitochondrial [Ca²⁺] in neuronal mitochondria. In the soleus fibres in the present study, the relatively rapid reversal of the mitochondrial rhod-2 signal in soleus fibres immediately after the end of stimulation suggests that the majority of the Ca²⁺ exists in the free form in the matrix of the mitochondria. On balance, it seems probable that the decline reflects increased activity of the Na⁺-Ca²⁺ exchange stimulated by the increased intracellular Na⁺ that occurs during repeated tetani [39]. CGP-37157, which inhibits the mitochondrial Na⁺-Ca²⁺ exchanger, both increased peak mitochondrial Ca²⁺ during the series of repeated tetani and slowed the rate of extrusion of Ca²⁺ after the stimulation had stopped (see Figure 2A and Figure 3A). CGP-37157 has a protective effect in neuronal excitotoxicity by blocking voltage-gated, including L-type, Ca²⁺ channels [40]. However, while L-type voltage-gated Ca²⁺ channels are present in both neurons and muscle, they differ in structure [41] and no evidence that CGP-37157 affects L-type Ca²⁺ channels in skeletal muscle has been reported (e.g., [30]).

One interesting finding of this study is the accelerated force decline observed during the induction of fatigue in the presence of CGP-37157. Tetanic force generation is governed by the amount of calcium released from the sarcoplasmic reticulum (SR). Since we demonstrate an increase in mitochondrial Ca²⁺, it can be asked if this reduces the SR-releasable Ca²⁺. Previous studies in rat cardiomyocytes [42] and skeletal muscle [43] have estimated that mitochondrial Ca²⁺ uptake is small and even during repeated contractions is not more than 1% of the total amount of SR Ca²⁺ released into the cytosol [30]. Thus, it appears unlikely that Ca²⁺ accumulation in the mitochondria can explain the decline in tetanic force.

Previously, Madsen et al. [28] reported that the elevations in mitochondrial Ca²⁺ induced by exhaustive exercise persisted for at least 60 min after the end of exercise in human muscle. In contrast, the present study finds that mitochondrial Ca²⁺ returned to control levels within 20 min of the end of repetitive activity. It should be noted that in the earlier study on human muscle, total mitochondrial Ca²⁺ was measured [28] while the rhod-2 used in the present study reports only the presence of free Ca²⁺ and not Ca²⁺ complexed with ions such as phosphate or bound to mitochondrial buffering proteins such as calmitine.

Mitochondrial Ca²⁺ uptake is implicated in both dissipation of the mitochondrial membrane potential and augmented ROS production and it is widely accepted that excessive ROS production exerts deleterious effects on cellular function [24,25,44]. Several groups have pointed out that ROS production can be considered physiologically vital as it is needed both to modify force production and modulate expression of genes involved in membranes and structural protein remodelling and metabolism [21,23,45]. Indeed, in the rat, strenuous exercise has little effect on ROS production in the soleus muscle [46]. In the present study, there were no differences in mitochondrial ROS production between soleus fibres exposed only to the Tyrode solution and those exposed to the Na⁺-Ca²⁺ exchange inhibitor, CGP-37157. This suggests that mitochondrial Ca²⁺ is of little importance in ROS generation in soleus fibres.

Little change in the mitochondrial membrane potential was observed after induction of fatigue under control conditions or exposure to CGP-37157. This is in line with results of a previous study on fast-twitch muscle fibres showing that there was no change in mitochondrial membrane potential in association with mitochondrial Ca²⁺ uptake in fast-twitch muscle fibres [20]. Thus, mitochondrial Ca²⁺ has little or no effect on mitochondrial potential possibly due to cations such as chloride accompanying Ca²⁺ entry into the mitochondria, although this needs to be confirmed by further research.

Study Limitations

Caution must be used in extrapolating these results. First, rhod-2 is a non-ratiometric indicator and this limits our knowledge as to the exact magnitude of the increase in mitochondrial Ca²⁺. Second, the study examined only soleus fibres from NMRI mice without taking into consideration whether they were oxidative (type I) or oxidative-glycolytic (type IIb) fibres. It is not certain that glycolyic type IIb or type IIx fibres would behave the same. Third, CGP-37157 was used as a “gold-standard” inhibitor of the mitochondrial Na⁺-Ca²⁺ exchanger. However, CGP-37157 has been reported to affect other ion channels and Ca²⁺ pumps and exchangers. While resting tension and tetanic force was unchanged after the 15 min equilibration period, subtle non-specific off-target effects of CGP-37157 cannot be entirely ruled out.

5. Conclusions

In conclusion, during extended periods of contractile activity in soleus fibres, mitochondrial Ca²⁺ is tightly regulated and exerts a stimulatory role in mitochondrial respiration. In the presence of CGP-37157, there is increased Ca²⁺ accumulation in mitochondria which results in impaired skeletal muscle force production, although the precise mechanism remains to be elucidated.