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Decrease in resting calcium and calcium entry associated with slow-to-fast transition in unloaded rat soleus muscle¹

Sezione di Farmacologia, Dipartimento Farmaco-Biologico, Università degli Studi di Bari, Via Orabona 4–Campus, Bari, Italy

²Correspondence: Sezione di Farmacologia, Dipartimento Farmaco-Biologico, Università degli Studi di Bari, Via Orabona 4—Campus, 70125, Bari, Italy. E-mail: conte{at}farmbiol.uniba.it

SPECIFIC AIMS

To assess the participation of the resting free cytosolic calcium ([Ca²⁺]_i) in the loading-dependent modulation of skeletal muscle phenotype, we measured in parallel resting [Ca²⁺]_i, voltage threshold for contraction, and caffeine-induced calcium release in soleus muscle fibers from control and unloaded rats. To further determine the mechanisms by which loading regulates [Ca²⁺]_i, we also measured and characterized the sarcolemmal permeability to calcium ions (SP-Ca) in extensor digitorum longus (EDL) and soleus muscle fibers of rats in control-bearing conditions and subjected to 3 and 14 days hind-limb unloading (HU).

PRINCIPAL FINDINGS

1. Resting [Ca²⁺]_i is muscle type-specific and is decreased in soleus muscle during HU
In control rats, the resting [Ca²⁺]_i in soleus muscle fibers was significantly higher than in EDL muscle as assessed by means of the fura-2 fluorescent calcium probe. After 3 days of HU, resting [Ca²⁺]_i was significantly decreased in soleus muscle fibers as compared with control. Prolonging suspension to 14 days further decreased soleus muscle resting [Ca²⁺]_i, which became similar to that measured in control EDL muscle fibers. Accordingly to these results, we found that by using a two-points voltage-clamp technique, soleus muscle had a lower voltage threshold for contraction than EDL muscle, but this difference was progressively reduced during suspension (Fig. 1B ).

View larger version (34K): [in this window] [in a new window]   Figure 1. Characterization of resting Mn2+ influx in soleus and EDL muscle fibers of control rats and in soleus muscle fibers of 3 (HU3d)- and 14 (HU14d)-day HU rats. A) Each bar represents the mean ± SEM value of mean-resting-quench rates measured in n fibers from N rats. B) Correlation between resting calcium level and quench rate in soleus muscle fibers during HU. C) Each bar represents the mean ± SEM value of quench rate before (solid bars) and after (open bars) application of Gd3+ determined in n fibers from N rats. , Significantly different from quench rate determined in absence of Gd3+, paired Student’s t-test, P < 0.02, for all groups. When difference is significant between groups, the level is indicated (first term in absence of Gd3+, second term in presence of Gd3+). D) Application of nifedipine induced an increase of the quench rate in all muscle fiber groups, P < 0.03, paired Student’s t-test. Each bar represents the mean ± SEM value of n fibers from N rats. *, Significantly different from value obtained in control soleus muscle fibers ANOVA with Bonferroni’s test. A, C) Statistical differences between quench rate of the different groups were determined using an ANOVA with Bonferroni’s test. Statistical difference versus: a, control soleus muscle fibers; b, soleus muscle fibers from HU3d rats; and c, soleus muscle fibers from HU14d rats. n.s., Nonsignificantly different.

2. Resting [Ca²⁺]_i decrease precedes slow-to-fast shift transition of soleus muscle phenotype and is not correlated to atrophy induced by HU
As a result of HU-induced atrophy, the fiber diameter and muscle-to-body weight of soleus muscle were significantly decreased during suspension, but these changes were not correlated to resting [Ca²⁺]_i decrease. In control conditions, that slow-twitch soleus muscle fibers were more sensitive (lower threshold); and more responsive (higher calcium transient amplitude) to caffeine than those of fast-twitch EDL muscle. After 3 days of suspension, little change was observed. In contrast, in soleus muscle of rats suspended for 2 weeks, the caffeine dose-response curve was significantly shifted toward higher caffeine concentrations, and the calcium transient amplitude obtained in response to 40 mM caffeine was reduced twofold as compared with control soleus, both being similar to that of control EDL muscle.

3. Resting skeletal muscle SP-Ca is dependent on resting tension and muscle type and is decreased in soleus muscle during suspension in correlation to resting [Ca²⁺]_i
To determine the resting tension-dependence of calcium influx, we measured the manganese-induced fura-2 quenching at different ranges of sarcomere length (SL; in µm: 1.8SL< 2.3; 2.3SL<2.6; and 2.6SL< 3.0). The SL/quench-rate relationships of all four muscle groups were bell-shaped, and the maximum quench-rate value was obtained for SL physiological range (2.5 µm). When muscle fibers were stretched until this SL value or above, quench rates were found to differ as follows: Control soleus > 3 days HU soleus > 14 days HU soleus > EDL muscles. Thus muscle fibers were stretched to set a SL value of 2.5 µm before all experiments. In these conditions, quench rate in resting control soleus muscle fibers was about twice that of EDL muscle. The mean quench-rate value in soleus muscle fibers was decreased by 30% after 3 days of HU (Fig. 1 A) and further decreased by 55% after 14 days of HU, becoming similar to that measured in control EDL muscle. A high linear correlation was found between mean quench-rate values and resting [Ca²⁺]_i of soleus muscle fibers from control and suspended rats (Fig. 1B ). These results were not dependent on difference in muscle fiber diameters as a result of muscle types or HU-induced atrophy.

4. Muscle specificity and HU-induced shift of SP-Ca are linked to difference in stretch-activated channels
As soleus muscle fiber quench rate was dependent on the resting tension, we assessed the participation of stretch-activated channels (SAC) to Mn²⁺ influx using Gd³⁺. Application of 50 µM Gd³⁺ reduced fluorescence quench in all muscle fiber groups. This block was more pronounced in control soleus muscle fibers than in EDL (Fig. 1C ). Moreover, during suspension, the inhibition of quench rate by Gd³⁺ was progressively decreased in soleus muscle fibers toward value observed in EDL muscle. Additionally, the persisting quench rates after Gd³⁺ application were similar between muscle fiber groups. As Gd³⁺ block was only partial, we used nifedipine to determine the relative involvement of leak and voltage-dependent calcium channels. In all muscle fiber groups, application of 25 µM nifedipine induced a significant increase of the quench rate, whereas inhibition of Mn²⁺ influx was never observed. This indicates the contribution of leak calcium channels to Mn²⁺ influx but excludes involvement of voltage-dependent ones. The nifedipine-induced quench-rate increase was lower in control soleus muscle than EDL ones (Fig. 1D ). During suspension, the nifedipine effect became similar in soleus and EDL muscles, without a shift in the absolute quench-rate increase induced by application of nifedipine.

CONCLUSIONS

After 3 days of suspension, resting [Ca²⁺]_i is already decreased in a drastic manner in soleus, but in this muscle, the functional characteristics are still typical of a slow-twitch muscle and immunostaining, and reverse transcriptase-polymerase chain reaction experiments reveal no change in myosin heavy chain protein and mRNA expression. Hence, resting [Ca²⁺]_i changes appear to precede most of the functional modifications resulting from HU-induced slow-to-fast transformation. Thus, the resting [Ca²⁺]_i decrease observed in soleus muscle as a result of HU may play an important role in triggering the slow-to-fast transition. Conversely, the resting [Ca²⁺]_i value is also a signature of muscle phenotype. Accordingly and in parallel to the slow-to-fast transformation of soleus muscle fibers, the resting [Ca²⁺]_i was further reduced by 14 days of HU. It is interesting that we found that resting [Ca²⁺]_i decrease was not correlated to HU-induced atrophy of soleus muscle. These data are in line with other studies, suggesting that atrophy of unloaded soleus muscle is mainly calcium-independent and results from the activation of the ubiquitin proteolytic pathway. In summary, resting [Ca²⁺]_i appears to be a crucial parameter for the HU-induced slow-to-fast transition of soleus muscle fibers, especially during the first days of suspension but would be in turn influenced by the functional changes.

One major finding of the present study was that calcium entry through the sarcolemma is greater in the slow-twitch soleus muscle fibers than in the fast-twitch EDL ones. During suspension, the passive manganese influx in soleus muscle fibers became similar to those of EDL ones after 2 weeks. We show that the difference in calcium permeability between the two muscles mainly arises from a higher expression and/or activity of SAC, which are gradually reduced as a result of suspension, contrary to the activity of the leak channels that remain unchanged.

The fact that passive calcium influx appears to be maximum when the muscle fibers are stretched close to in vivo resting conditions suggests that the role of this calcium influx could be related to long-lasting, resting regulation of muscle, such as gene transcription regulation rather than short-term regulation of contractile properties. In addition, reduction of SP-Ca in unloaded soleus muscle is an early event and is tightly correlated to the resting calcium decrease. We cannot exclude that resting [Ca²⁺]_i decrease depends on various parameters, in particular, up-regulation of sarcoplasmic reticulum Ca²⁺-ATPase activity. Alternatively, as soleus muscle has been shown to be shortened in vivo during suspension, this could induce a reduced SAC activity, resulting in early decrease of calcium influx and a consequent decrease of resting [Ca²⁺]_i. This could be involved in the initial events of the slow-to-fast transition, via modulation of Ca²⁺-dependent pathways that could further decrease resting [Ca²⁺]_i and SP-Ca as a result of the slow-to-fast transition of calcium-handling mechanisms (Fig. 2 ). If confirmed, SAC could constitute mechanisms by which skeletal muscle fibers could sense loading conditions. Pharmacological tools able to modulate calcium homeostasis and SAC activity may be of potential interest to counteract disuse-induced muscle alterations occurring in microgravity conditions or as a result of limb immobilization after spinal cord injury or prolonged bed resting.

View larger version (29K): [in this window] [in a new window]   Figure 2. Schematic diagram of the proposed intracellular pathway by which soleus muscle senses unloading and undergoes consequent slow-to-fast phenotype transition. From the beginning of HU, soleus will be shortened, inducing a decrease of SAC activity and a consequent reduction of resting calcium influx. In turn, resting [Ca2+]i will slowly decrease, which constitutes the first step of the slow-to-fast transition. This process may be achieved through a reduced activity of calcineurin, a calcium-dependent phosphatase that is known to play an important role in the maintenance of slow-twitch phenotype. During HU, soleus muscle fibers will progressively acquire calcium-handling mechanisms typical of a fast-twitch muscle, such as the fast isoform of the sarcoplasmic reticulum Ca2+-ATPase pump. Also, SAC activity will be further decreased. All during the process, resting [Ca2+]i will be gradually decreased toward a value typical of a fast-twitch muscle, maintaining the corresponding muscle phenotype.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1012fje; doi: 10.1096/fj.02-1012fje

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