Dihydropyridine Receptor Inhibition Attenuates Force and Fiber Cross-Sectional Area Decrease in the Three-Day Unloaded Rat Soleus Muscle

Abstract

The depolarization of the sarcolemma is one of the first effects of unloading on skeletal muscle. We hypothesized that unloading-induced activation of the dihydropyridine receptor (DHPR), a voltage-sensitive L-type Ca²⁺ channel, and depolarization of the sarcolemma trigger intracellular Ca²⁺ release from the sarcoplasmic reticulum and activation of Ca²⁺-dependent signaling pathways, resulting in muscle atrophy. Nifedipine, a DHPR calcium channel blocker, was used to study the role of DHPR in the regulation of signaling pathways during three days of rat soleus muscle unloading/hindlimb suspension. Inhibition of the DHPR during unloading attenuates the decrease in soleus muscle contractile properties, prevents the accumulation of ATP, ROS, and Ca²⁺ content in the sarcoplasm and the mitochondria, and blocks the decrease in PGC1alpha mRNA expression and Junctophilin-1 (JP1) proteolysis. In nifedipine-treated rats, the improvement of the unloaded soleus muscle contractile properties could be mediated by blocking the calpain-mediated degradation of the cytoskeletal proteins. DHPR blocking could be one of the future directions for the preservation of contractile properties of inactive/unloaded muscle.

1. Introduction

Skeletal muscle is critically important for total body function and metabolism. Skeletal muscles do more than just maintain posture/position, stabilize joints, and enable movement; they also act as a vital metabolic hub by consuming glucose to stabilize blood sugar levels. Emerging research indicates that these muscles function as endocrine organs, releasing bioactive myokines and exerkines that coordinate the health of nearly every tissue in the body [1]. Skeletal muscle atrophy may arise from a variety of pathological and physiological conditions, including prolonged immobilization, malnutrition, chronic illness, and aging [2].

Skeletal muscle unloading leads to atrophy due to the imbalance between protein synthesis and protein degradation rates [3,4]. Changes in the intracellular signaling pathways start during the first few hours to days after muscle unloading [5]. Depolarization of the sarcolemma due to the inactivation of the alpha2 subunit of Na, K-ATPase, is one of the first effects of unloading, and it precedes skeletal muscle atrophy [6]. We hypothesized that activation of the DHPR voltage-sensitive L-type calcium (Ca²⁺) channel and depolarization of the sarcolemma during unloading trigger intracellular Ca²⁺ release from the sarcoplasmic reticulum and activation of Ca²⁺-dependent signaling pathways.

Skeletal muscle atrophy and a decrease in muscle mass occur rapidly after muscle unloading caused by prolonged bed rest during hospitalization, microgravity, or experimentally created similar conditions [7,8]. Some studies report logarithmic loss of strength and atrophy of weight-bearing muscles after unloading [9]. The fastest rate of muscle force loss is observed during the early stages of bed rest, and later it reaches a plateau [9,10]. Even a short-term hospitalization (≤5 days) that includes inactivity/immobilization is sufficient to significantly decrease muscle mass and cause a decrease in maximum muscle strength [9]. In humans, after two weeks of bed rest, muscle strength declines much faster than muscle atrophy [9]. Multiple studies previously reported unloaded muscle atrophy, loss of strength, and increased fatigue in rodents [3,11,12]. Unloading-induced changes in human skeletal muscle correlate well with the observations in rodents; therefore, rodents are a good model for these studies.

It has been reported that about 79% of muscle strength loss can be explained by muscle atrophy, while the remainder is most likely to be due to changes in the mechanical properties of individual muscle fibers, excitation–contraction coupling, fiber architecture, destruction of cytoskeletal proteins, tendon stiffness, muscle denervation, damage to neuromuscular junctions, and supraspinal changes [9]. The authors note that over time, bedrest resulted in a greater magnitude of change for isometric muscle force than for muscle atrophy [9]. To summarize, 79% of muscle strength loss can be explained by muscle atrophy, while the other 21% is attributed to the less-described effects of unloading.

The maximum decrease in strength occurs at the earliest stages of muscle unloading, and these changes are the least studied. To develop a method for the prevention of strength loss during muscle unloading, it is required to elucidate the mechanisms of its initiation. To study the mechanisms of these early processes and to identify ways to prevent the loss of muscle strength, we evaluated the effect of the L-type calcium channel blocker nifedipine in a rat model during three days of unloading using hindlimb suspension. The soleus muscle was selected since it bears the maximum weight load and undergoes the fastest changes after unloading. It is known that after 3 days of soleus muscle unloading, there is an increased accumulation of Ca²⁺ in the sarcoplasm [13]. Previous studies using indirect methods of Ca²⁺ content evaluation showed that both systemic and local application of nifedipine prevented the accumulation of Ca²⁺ in muscle fibers during unloading [14]. Nonetheless, muscle force was not evaluated, and the connection between the nifedipine-induced decrease in Ca²⁺ accumulation and the effect on muscle fiber force was not established.

We hypothesized that the nifedipine-mediated inhibition of the DHPR and prevention of membrane depolarization would prevent Ca²⁺ increase in unloaded soleus muscle. This will reduce muscle force loss and decrease muscle proteolysis. For example, the activation of the calcium-dependent calpains could be reduced. It was previously reported that three-day inhibition of calpains via blocking of DHPR channels prevented the destruction of cytoskeletal proteins during unloading [15]. Currently, there are no published studies evaluating the effects of nifedipine on the loss of muscle force during unloading. There is also no effective treatment for preventing loss of muscle force during the early stages of muscle unloading in patients with hypokinesia, acute hospitalization, and limb immobilization. If our hypothesis is correct, then lowering Ca²⁺ levels with nifedipine during unloading will prevent a decline in muscle contractile properties. This knowledge might help to develop an effective preventive measure for the early stages of hypokinesia in humans.

2. Results

2.1. The Effect of Nifedipine on Unloaded Soleus Muscle Mass and Cross-Sectional Area

After three days of unloading, there were no significant changes in the total body weight amongst all three experimental groups (Table 1). At the same time, soleus muscle mass was significantly lower in the 3HS (3-day hindlimb suspended) and 3HS+N (3-day hindlimb suspended treated with nifedipine) groups when compared with the control (C) (Table 1).

Table 1.

Effect of nifedipine on the total body weight and the weight of the unloaded soleus muscle.

	C	3HS	3HS+N
Total Body Weight (g)	203.7 ± 14.2	181.6 ± 19.7	178.2 ± 8.6
Soleus Muscle Weight (mg)	101.5 ± 3.8	78.0 ± 3.0 *	76.9 ± 1.7 *
Dry Soleus Muscle Weight Index	19.9 ± 0.75	15.8 ± 0.8 *	17.1 ± 0.22 *
Soleus Muscle Index (mg/g)	0.47 ± 0.02	0.42 ± 0.01 *	0.41 ± 0.01 *

* Indicates a significant difference from the C group (p < 0.05). Data are shown as mean value ± SD. n = 8 for each group.

The cross-sectional area (CSA) of slow muscle fibers in the 3HS and 3HS+N groups decreased by 24% and 12%, respectively, but these decreases did not reach statistically significant values compared with the control (Figure 1). In contrast, the decrease in the CSA of fast and hybrid muscle fibers in the 3HS group by 26% and 27%, respectively, was statistically significant when compared with the control. Nifedipine treatment diminished the CSA decline of fast and hybrid muscle fibers in the 3HS+N group (Figure 1). The decrease in CSA of fast and hybrid muscle fibers in the 3HS+N group was diminished by 17% and 12%, respectively, compared with the 3HS group. The CSA of fast and hybrid muscle fibers in the 3HS+N group was not statistically different from either the C or 3HS group (Figure 1).

Figure 1.

Evaluation of the CSA of slow (n: C = 4, 3HS = 6, 3HS+N = 5) (A), fast (n: C = 4, 3HS = 6, 3HS+N = 4) (B), and hybrid (n: C = 4, 3HS = 6, 3HS+N = 4) (F) muscle fibers and evaluation of the relative content of slow (C) (n: C = 4, 3HS = 6, 3HS+N = 5), fast (n: C = 4, 3HS = 6, 3HS+N = 5) (D), and hybrid (n: C = 4, 3HS = 6, 3HS+N = 4) (E) muscle fibers in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). * Indicates a significant difference, p < 0.05. Bar = 100 μm.

The percentage of slow muscle fibers in the 3HS+N group was significantly higher than in both the C and 3HS groups by 22% and 26%, respectively. At the same time, the percentage of fast muscle fibers in the 3HS+N group was significantly lower than in the C and 3HS groups by 31% and 42%, respectively (Figure 1).

2.2. The Effect of Nifedipine on Protein Synthesis and Protein Degradation Pathways in Unloaded Soleus Muscle

The protein synthesis rate was significantly lower in the soleus muscle of both unloaded groups (3HS and 3HS+N, Figure 2A) when compared with the control. There was also a lower level of phospho-S6 protein in both unloaded groups when compared with the control (3HS and 3HS+N, Figure 2B). Treatment with nifedipine did not prevent the unloading-induced upregulation of eEF2 protein phosphorylation in the soleus muscle of the 3HS+N group (Figure 2C). There were lower levels of 18S and 28S rRNA in both unloaded groups (3HS and 3HS+N, Figure 2D,E). In combination, these data suggest a decrease in anabolic processes in the unloaded soleus muscle of nifedipine-treated and untreated rats.

Figure 2.

Evaluation of the protein synthesis rate (n = 8 in each group) (A), phospho-S6 content (n: C = 7, 3HS = 8, 3HS+N = 8) (B), phospho-eEF2 content (n = 8 in each group) (C), 18S (n = 8 in each group) (D), and 28S (n = 8 in each group) (E) rRNA in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). * Indicates a significant difference, p < 0.05.

The evaluation of the ubiquitin-proteasomal signaling pathway showed that nifedipine did not affect the unloading-induced mRNA expression of ubiquitin, MuRF1, and MAFbx (3HS and 3HS+N, Figure 3A–C).

Figure 3.

Evaluation of ubiquitin (A) (n = 8 in each group), MuRF1 (B) (n = 8 in each group), and MAFbx (C) (n = 8 in each group) mRNA expression in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). * Indicates a significant difference, p < 0.05.

2.3. The Effect of Nifedipine on Calcium Signaling Pathways in Unloaded Soleus Muscle

Unloading resulted in an increase in the Ca²⁺ levels in the sarcoplasm (Oregon Green BAPTA-1; OGB-1) and mitochondria (Rhod-2 AM) of the unloaded soleus muscle (3HS group, Figure 4A,B). Treatment with nifedipine prevented the unloading-induced upregulation of the Ca²⁺ levels (3HS+N group, Figure 4A,B). Two different methods for evaluating Ca²⁺ levels using OGB-1 and Rhod-2 AM (Figure 4A,B) showed similar results.

Figure 4.

Evaluation of Ca²⁺ levels in the sarcoplasm (Oregon Green, (A) (n = 8 in each group) and mitochondria (Rhod-2 AM, (B) (n: C = 7, 3HS = 7, 3HS+N = 8) in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). * Indicates a significant difference, p < 0.05. Bar = 100 μm.

The evaluation of CaMK IIb phosphorylation showed a significant unloading-induced increase in soleus muscle compared with the control (Figure 5A). In the nifedipine-treated rats, the phosphorylation of CaMK IIb was not statistically different from the control values (Figure 5A).

Figure 5.

Evaluation of phospho-CaMK IIb (A) (n: C = 7, 3HS = 6, 3HS+N = 7) and phospho-IP3R (B) (n: C = 7, 3HS = 6, 3HS+N = 6) in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). The content of phospho-CaMK IIb was normalized to the content of total CaMK IIb, and the content of phospho-IP3R was normalized to the content of total IP3R. * Indicates a significant difference, p < 0.05.

The content of phospho-IP3R in the nuclear fraction of soleus muscle fibers was significantly increased in the unloaded soleus muscle compared with the control (3HS, Figure 5B). Treatment with nifedipine prevented this increase (3HS+N, Figure 5B).

2.4. The Effect of Nifedipine on the Contractile Properties, Oxidative Stress, and Energy-Related Signaling Pathways in Unloaded Soleus Muscle

Unloading resulted in a significant decrease in the muscle fiber physiological cross-sectional area of the soleus muscle (Table 2). Nifedipine treatment did not prevent an unloading-induced decrease in the muscle fiber physiological cross-sectional area (Table 2). Despite this, only the unloaded soleus muscle without nifedipine treatment had a significant decrease in the maximum force of the single isometric contraction and the maximum force of the tetanic contractions compared with the soleus muscle of control rats (Table 2). Maximum specific force of single isometric contraction and maximum specific force of maximum contractions did not differ between the 3HS and C groups, but in the 3HS+N group, the maximum specific force of a single isometric contraction was significantly higher than in the 3HS group, and the maximum specific force of tetanic contractions in the 3HS+N group was significantly greater than in both the C and 3HS groups (Table 2).

Table 2.

Effect of nifedipine on the contractile properties of unloaded soleus muscle.

	C	3HS	3HS+N
Soleus muscle length (mm)	20.2 ± 0.2	19.6 ± 0.3	19.4 ± 0.3
Soleus muscle physiological cross-sectional area (mm2)	5.1 ± 0.2	3.8 ± 0.1 *	4.2 ± 0.3 *
The maximum force of the single isometric contraction (mN)	99.4 ± 7.8	75.6 ± 3.9 *	88.7 ± 10.2
The maximum force of the tetanic contractions (mN)	656.1 ± 31.4	496.1 ± 48.6 *	528.1 ± 61.6
Specific maximum force of the single isometric contraction (mN/mm2)	22.98 ± 3.0	18.53 ± 2.2	26.79 ± 7.8 #
Specific maximum force of the tetanic contractions (mN/mm2)	127.9 ± 15.01	116.3 ± 27.8	160.2 ± 22.85 *#

* Indicates a significant difference from the C group (p < 0.05), and # indicates a significant difference from the 3HS group. N = 8 for each group. Data are shown as mean value ± SD.

There was a significant increase in the ATP content in the soleus muscle after unloading (3HS group, Figure 6A). Treatment with nifedipine blocked this increase (3HS+N group, Figure 6A). In contrast, the content of phospho-AMPK was significantly decreased in the unloaded soleus muscle when compared with the control (3HS group, Figure 6B). The content of phospho-AMPK in the unloaded soleus muscle of nifedipine-treated rats was not statistically different from the control (3HS+N group, Figure 6B).

Figure 6.

Evaluation of ATP (A) (n: C = 7, 3HS = 7, 3HS+N = 7) and phospho-AMPK (B) (n: C = 6, 3HS = 6, 3HS+N = 6) content in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). The content of phospho-AMPK was normalized to the content of total AMPK. * Indicates a significant difference, p < 0.05.

In skeletal muscle, Junctophilin-1 (JP1) couples sarcolemmal and intracellular calcium channels [16]. After three days of unloading, the content of JP1 in the 3HS group decreased by half compared to the content in the control group (Figure 7A). The content of JP1 in the nifedipine-treated group was not significantly different from the control (3HS+N group, Figure 7A).

Figure 7.

Evaluation of JP1 (A) (n: C = 7, 3HS = 7, 3HS+N = 7), DHE IF (n: C = 7, 3HS = 7, 3HS+N = 7) (B), and PGC1alpha mRNA (n = 8 for each group) (C) content in soleus muscles of control rats (C), rats with 3 days of unloading (3HS), and 3 days of unloading treated with nifedipine (3HS+N). * Indicates a significant difference, p < 0.05.

Evaluation of the reactive oxygen species (ROS) using DHE showed a significant increase in the unloaded soleus muscle when compared with the control (3HS group, Figure 7B). Nifedipine treatment blocked this increase (3HS+N group, Figure 7B).

PGC1alpha mRNA content was significantly decreased in the 3HS group (Figure 7C). This decrease was prevented by nifedipine treatment (3HS+N group, Figure 7C).

3. Discussion

Our current findings validate previous observations by providing direct measurements of intracellular Ca²⁺ concentrations within isolated muscle fibers. Crucially, while earlier work hinted at this relationship, it did not assess muscle force. This study, therefore, establishes the previously unconfirmed link between the nifedipine-induced reduction in Ca²⁺ accumulation and its direct functional effect on muscle fiber force generation. Unloading results in progressive skeletal muscle atrophy. In rats, three days of unloading is the earliest time point for a detected decrease in soleus muscle weight [5]. In the current study, the wet and dry mass of the unloaded soleus muscle significantly decreased in rats with and without nifedipine treatment. The CSA of fast and hybrid fibers was significantly reduced only in the 3HS group relative to the control group. There was no significant decrease in the CSA of slow fibers in either of the unloaded groups. The differences in the dry weight loss and CSA of slow fibers between the 3HS and 3HS+N groups may be related to differences in water content. It is known that during unloading, there are changes in the fluid distributed throughout the body due to changes in renal filtration [17]. In the current study, we observed that the prevention of slow-to-fast muscle fiber transition in the 3HS+N group could contribute to the functional properties of the soleus muscle in this group.

3.1. The Effect of Nifedipine on the Anabolic and Catabolic Signaling Pathways in Unloaded Soleus Muscle

In this study, we detected that a decrease in the dry weight of the unloaded soleus muscle correlates well with the unloading-induced decrease in protein synthesis rate in rats with and without nifedipine treatment. The content of phospho-S6 protein and the expression of 18S and 28S rRNA were also decreased in both unloaded rat groups. In eukaryotes, 18S rRNA is a component of the small ribosomal subunit (40S). In addition, 28S rRNA catalyzes peptide bond formation and is a component of the large ribosomal subunit (60S). The 18S and 28S RNAs are markers of the ribosomal content in skeletal muscle [18]. The decrease in the phospho-S6 protein, and 18S and 28S rRNAs indicates a decline in muscle anabolic activity. The results of the current study correlate well with the previously published data showing a decrease in 18S and 28S rRNA content in the unloaded soleus muscle [19].

Interestingly, eEF2K phosphorylation of eEF2 inhibits protein translation [20]. In a previous study, the content of phosphorylated eEF2 increased in unloaded soleus muscle [21]. Similar changes were observed in the current study. Nifedipine treatment did not affect the unloading-induced increase in the content of phosphorylated eEF2.

The evaluation of the markers of the ubiquitin–proteasome signaling pathway showed a significant increase in the expression of mRNA of the E3 ligases MuRF1, MAFbx, and ubiquitin in both unloaded rat groups relative to the control group. Nifedipine treatment did not prevent the increase in the mRNA expression of E3 ligases.

The results of the current study showed that administration of nifedipine during unloading does not prevent the atrophy of the rat soleus muscle. This observation is supported by the high level of expression of E3 ligases and the reduced content of markers of the anabolic signaling pathway in the soleus muscle of both unloaded groups.

3.2. The Effect of Nifedipine on the Contractile Properties of Unloaded Soleus Muscle

Muscle atrophy and the decrease in muscle contractile properties start early after inactivity or unloading. For the rat soleus muscle, muscle atrophy and the decrease in muscle strength can be detected at three days of unloading. The degree of this decrease was higher for muscle atrophy than for a decline in muscle contractile properties [9]. In the current study, there was a significant decrease in soleus muscle contractile properties at three days of unloading. The treatment with nifedipine prevented this decrease. This suggests that DHPR calcium channels are involved in the regulation of muscle contractile properties.

In humans, the decline in muscle strength is observed after 5 to 14 days of bed rest-induced inactivity [9]. This happens before the detection of any signs of muscle atrophy. The authors stated that the decline in muscle strength could not be completely described by the decreased content of contractile proteins [9]. Similarly, the difference in the decline in the unloaded muscle contractile properties in the 3HS and 3HS+N groups observed in the current study could not be completely explained by differences in muscle atrophy. The variations in the fast fibers’ cross-sectional areas and in the number of hybrid fibers between these two groups could contribute to the observed differences in the contractile properties. The observed reduction in myoplasm Ca²⁺ concentration in the 3HS+N group compared to the 3HS group likely contributes to nifedipine’s protective effect on specific muscle force. This mechanism potentially involves the mitigation of Ca²⁺-dependent calpain activation, thereby preventing the subsequent proteolytic degradation of essential cytoskeletal proteins.

The current study detected differences in the sarcoplasmic Ca²⁺ between the 3HS and 3HS+N groups. Increased sarcoplasmic Ca²⁺ concentration in the 3HS group could have stimulated the increase in the proteolysis of the cytoskeletal and contractile proteins. It was previously reported that the unloading-induced increase in the sarcoplasmic Ca²⁺ concentrations activated calcium-dependent proteases, calpains [14]. Calpain inhibition prevented the proteolysis of the cytoskeletal proteins [22].

The content of JP1 was significantly decreased in the unloaded soleus muscle. This decrease was prevented by nifedipine treatment. JP1 plays an important role in muscle contraction by the mechanical stabilization of the transverse tubular system and SR membranes [23,24]. Increased sarcoplasmic Ca²⁺ induces Ca²⁺-dependent degradation of JP1 by calpains, and this could result in muscle weakness [23,25]. The decreased content of JP1 in unloaded soleus muscle correlates well with the increased sarcoplasmic Ca²⁺ content in this group and previously reported activation of calpains [26]. Therefore, Ca²⁺-induced activation of calpains and calpain-mediated degradation of JP1 could be one of the contributing factors to the decline in contractile properties in unloaded soleus muscle in the current study.

3.3. The Effect of Nifedipine on the Content of ATP in the Unloaded Soleus Muscle

The content of ATP was significantly increased in the unloaded soleus muscle, while treatment with nifedipine blocked this increase. The unloading-induced increase in ATP content in the soleus muscle was previously reported [27]. At the same time, the content of phospho-AMPK in unloaded muscle was significantly decreased, and nifedipine treatment diminished this decrease. AMPK is one of the key regulators of energy homeostasis. Therefore, inhibition of the DHPR with nifedipine has a significant effect on muscle energy metabolism by preventing ATP increase and affecting sarcoplasmic Ca²⁺ content.

3.4. The Effect of Nifedipine on the Content of Ca²⁺ in the Unloaded Soleus Muscle

Sarcoplasmic Ca²⁺ content was significantly increased in the unloaded soleus muscle. Nifedipine treatment prevented this increase. The unloading-induced increase in sarcoplasmic Ca²⁺ content in the soleus muscle was previously reported [28]. The phosphorylation of CaMK IIb was also increased by unloading, and this increase was prevented by nifedipine treatment. CaMK is a marker of the Ca²⁺-dependent activation of signaling pathways. Intracellular Ca²⁺ is one of the regulators of CaMK activity [29]. CaMK regulates the phosphorylation/activity of many muscle proteins, including AMPK [29] and several transcription factors [30,31].

It was previously reported that ATP can upregulate Ca²⁺ content in soleus muscle fibers by activation of P2Y2 receptors and downstream IP3R signaling [32]. In the current study, the content of nuclear IP3R was increased in the unloaded soleus muscle, while treatment with nifedipine prevented this increase. IP3R are Ca²⁺-dependent receptors that are activated and translocated into nuclei by increased cytoplasmic Ca²⁺ concentrations [33].

The results of the current study suggest that DHPR inhibition with nifedipine affects sarcoplasmic Ca²⁺ content in muscle fibers, CaMK phosphorylation, and regulation of Ca²⁺-dependent signaling pathways in unloaded soleus muscle. These changes are apparently associated with the prevention of an increase in cytoplasmic Ca²⁺ levels in unloaded muscles of rats treated with nifedipine. Moreover, the accumulation of Ca²⁺ in the myoplasm results in upregulated ROS concentration that further increases the leakage of Ca²⁺ from the sarcoplasmic reticulum and loss of muscle force [34,35].

3.5. The Effect of Nifedipine on the ROS and PGC1alpha in the Unloaded Soleus Muscle

The content of ROS was significantly upregulated in the three-day unloaded soleus muscle, while blocking the increase in sarcoplasmic Ca²⁺ content with nifedipine prevented ROS accumulation. Mitochondria serve as the primary source of ROS concentrations; specifically, mitochondrial Ca²⁺ overload acts as a critical trigger for augmented ROS production [36,37]. There was a significant increase in the content of Ca²⁺ in the mitochondria of the 3HS group, but not in the 3HS+N group, when compared with the control group. Our findings suggest that DHPR inhibition in the unloaded soleus muscle may attenuate ROS production by preventing mitochondrial Ca²⁺ overload.

Ca²⁺ transfer between the endoplasmic reticulum (ER) and mitochondria is accomplished by Ca²⁺ transport systems located on mitochondria associated with ER membranes [38]. Intramitochondrial Ca²⁺ levels are intrinsically linked to cytosolic Ca²⁺ concentrations. Some authors propose that mitochondria function as calcium sinks, internalizing excessive cytosolic Ca²⁺ when Ca²⁺ levels in the sarcoplasm are elevated [39]. Therefore, DHPR inhibition could lead to an intramitochondrial Ca²⁺ decrease, preventing Ca²⁺ accumulation in the myoplasm. To the best of our knowledge, the present study is the first to demonstrate that DHPR calcium channel inhibition effectively attenuates mitochondrial Ca²⁺ accumulation during skeletal muscle unloading.

Elevated oxidative stress and ROS accumulation are known to trigger the activation of calcium-dependent calpains in skeletal muscle [40]. Once activated, calpains can disrupt cytoskeletal integrity, leading to reduced muscle stiffness and impaired force generation [41]. Furthermore, oxidative stress suppresses protein synthesis while accelerating proteolysis through the induction of atrogene expression [40]. ROS may also directly oxidize sarcomeric proteins, thereby decreasing the calcium sensitivity of the actomyosin complex and reducing maximal contractile force [42]. At the same time, it has been previously reported that oxidative stress is not a major cause of disuse muscle atrophy [43].

PGC1alpha is a major regulator of mitochondrial content in skeletal muscle [44]. The current study reports a decrease in PGC1alpha mRNA expression in the unloaded soleus muscle. These results correlate well with previous observations [43]. It was reported that PGC1alpha regulates fiber type transition during skeletal muscle unloading [45]. Changes in the composition of the muscle fibers in the unloaded soleus muscle could be related to the level of PGC1alpha expression. Furthermore, high-energy phosphates block the mitochondrial biogenesis markers’ expression, including PGC1alpha, and slow myosin expression in unloaded soleus muscle [46]. The results of the current study confirm previously reported data.

One possible hypothesis for the regulation of cellular signaling during functional unloading is the following: sarcolemmal depolarization during muscle unloading results in the opening of DHPR channels. DHPR channels are known to be closely associated with Pannexin 1 (Panx1) channels [47], which facilitate the efflux of sarcoplasmic ATP into the extracellular space. We previously demonstrated that blocking Panx1 with probenecid during muscle unloading leads to increased sarcoplasmic ATP accumulation, likely due to the inhibition of ATP efflux [48]. While DHPR inhibition also stabilizes intracellular macroergic phosphate levels, this effect appears independent of Panx1 activity. Instead, DHPR-mediated stabilization likely results from the normalization of synthesis and consumption pathways for high-energy phosphates within the muscle cells.

3.6. Study Limitations

Nifedipine is a medication used to treat high blood pressure and chest pain, and it was once one of the most widely used medications for hypertension. Side effects in humans include headache, flushing, nausea, heartburn, and constipation. The side effects in most people are not very severe or life-threatening. Nifedipine has been replaced by newer blood pressure medications. Although nifedipine could have had some effect on cardiac and smooth muscle, the short-term treatment used in the current study is unlikely to have severe negative effects on the experimental rats.

Another limitation is that this study has not evaluated the long-term effect of nifedipine on unloaded skeletal muscle. It is important to know whether the effects on muscle force will be preserved after a long-term muscle unloading. This is something that we plan to evaluate in the future.

Only the soleus muscle was evaluated in the current study. After three days of unloading, there were no significant changes in the size/weight of the gastrocnemius or tibialis muscle. When planning a long-term study, we will include the evaluation of the effects of nifedipine on other muscles.

The effect of nifedipine on muscle unloading was only tested in male rats. We expect that the effects in female rats will be similar, but in the future, this needs to be tested.

4. Materials and Methods

4.1. Animal Protocol Approval

This study was conducted at the Institute of Biomedical Problems (IBMP), Russian Academy of Sciences (RAS), in strict accordance with the ARRIVE guidelines [49] and established biomedical ethics standards [50]. The experimental protocols involving animals were reviewed and approved by the RAS Committee on Bioethics (Protocol No. 673, approved on 2 December 2024).

4.2. Animal Procedures

Twenty-four male Wistar rats (2 months old) were randomly allocated into three experimental groups (n = 8 per group): a control group (C), a 3-day hindlimb suspension/unloading group (3HS), and a 3-day hindlimb suspension/unloading group treated with the DHPR inhibitor nifedipine (3HS+N). Animals in the C and 3HS groups received daily intraperitoneal (i.p.) injections of a vehicle placebo consisting of 400 µL of 10% DMSO and 10% Tween 80 in physiological saline. The 3HS+N group received daily i.p. injections of nifedipine (Teva, Israel) at a dosage of 5 mg/kg body weight, dissolved in the same vehicle. This nifedipine dosage was selected based on previous optimization studies in both rats and cultured muscle cells [51,52]. A control group of unloaded rats treated with nifedipine was not used in this study since it does not represent an appropriate control for the unloaded nifedipine-treated group. The earliest effect observed within several hours of unloading was the depolarization of the sarcolemma due to the inactivation of Na, K-ATPase [53]. This is the main mechanism of membrane depolarization after unloading [53]. Depolarization precedes muscle atrophy, and it causes activation of pannexin channels, regulating ATP exit from the sarcoplasm [54,55]. Since the control group did not have depolarization of the sarcolemma or activation of pannexin channels, treatment of this group with nifedipine would cause processes that are not comparable to the mechanisms activated in the unloaded group treated with nifedipine.

Animals were subjected to hindlimb suspension using the Morey-Holton tail-casting method with Novikov’s modifications [56,57]. Briefly, animals were suspended by the tail using a swivel system and adhesive tape, maintaining the hindlimbs in an elevated, non-weight-bearing position. Rats were granted ad libitum access to food and water and could move freely within the cage using their forelimbs. At the conclusion of the study, rats were euthanized via an overdose of Avertin. The right soleus muscles were immediately excised for ex vivo contractile force assessment. The left soleus muscles were dissected and weighed; a ~5 mg specimen from the mid-belly region was utilized to determine the dry weight. The dry weight index was defined as the ratio of wet weight to dry weight. The soleus muscle weight index was calculated by normalizing the wet muscle mass to the total body weight of the animal. Remaining portions of the left soleus were flash-frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

4.3. Evaluation of Muscle CSA and Fiber Type Composition

Transverse soleus muscle sections (9 μm thick) were prepared using a Leica cryostat (Leica Biosystems, Germany), air-dried, and stored at −20 °C. For immunohistochemical analysis, sections were thawed and rehydrated in phosphate-buffered saline (PBS) for 20 min. Myofiber type distribution and cross-sectional areas (CSAs) were determined using an established protocol [58]. Sections were incubated with primary antibodies against slow MyHC I(β) (1:300, Sigma-Aldrich, St. Louis, MO, USA) and fast MyHCs (1:70, DSMZ, Braunschweig, Germany), followed by secondary antibodies Alexa Fluor 350 and Alexa Fluor 546 (1:1000; Molecular Probes, Eugene, OR, USA). Images were captured under standardized conditions with consistent exposure times, including negative controls to verify specificity. Using ImageJ software Java 8, fiber boundaries were manually traced to calculate the CSAs and fiber type proportions. A minimum of 100 fibers per sample were analyzed; samples with fewer than 100 fibers of a specific type (especially hybrid or fast-twitch) were excluded from that specific analysis.

4.4. Analysis of the Contractile Properties of the Soleus Muscle

Following dissection, isolated soleus muscles were equilibrated for 15 min in chilled Ringer–Krebs solution continuously aerated with carbogen (95% O₂/5% CO₂). During equilibration, silk sutures were secured to the tendons to facilitate mounting. The muscles were then transferred to a temperature-controlled testing chamber (37 °C) and positioned between a fixed hook and an Aurora Scientific 305C-LR Dual-Mode Lever System (Aurora Scientific, Aurora, ON, Canada). Optimal muscle length L₀ was established by delivering supramaximal square-wave pulses (0.5 ms, 20 V) and identifying the length that elicited maximal twitch force [59]; this length was verified using a digital caliper. Isometric tetanic force was subsequently assessed at L₀ via a 2 s stimulation train (1 ms pulses at 40 Hz). Specific force was determined by normalizing absolute values to the physiological cross-sectional area (pCSA), calculated from the wet muscle weight, L₀, and a muscle density of 1.07 g/cm³ [60]. Data acquisition was performed at 10 kHz using the Aurora Scientific 615A Analysis Software Suite.

4.5. Muscle ROS Content Evaluation

Superoxide production was assessed using dihydroethidium (DHE) staining, a well-established indicator of reactive oxygen species (ROS) in both in vitro [61] and in vivo [62]. Upon oxidation by superoxide, DHE intercalates with DNA to emit red fluorescence [61]. Transverse muscle sections (10 μm) were incubated with 5 μM DHE for 30 min at 37 °C in a dark environment. After rinsing with phosphate-buffered saline (PBS), sections were visualized via fluorescence microscopy. All photomicrographs were captured using standardized exposure settings, and negative controls were included to verify the specificity of the fluorescent signal.

4.6. Evaluation of the Protein Synthesis Rate

To evaluate the rate of protein synthesis, the SUnSET (Surface Sensing of Translation) method was employed as previously described [63,64]. Thirty minutes prior to muscle excision, rats received an intraperitoneal injection of puromycin (0.04 μmol/g body weight) dissolved in 100 μL of PBS. Subsequent puromycin incorporation into soleus muscle was quantified via Western blot analysis using anti-puromycin antibodies.

4.7. Western Blotting

Approximately 15 mg of tissue from the mid-belly of each soleus muscle was homogenized in RIPA lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA). Homogenates were centrifuged at 12,000× g for 15 min at 4 °C, and the resulting supernatant was collected for analysis. Total protein concentration was quantified using Bradford reagent and an Epoch spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA). Subsequent electrophoresis and Western blotting procedures followed previously established protocols [27].

The following primary antibodies were used:

• From Thermo Fisher Scientific, Waltham, MA, USA: phospho-CaMK II (Thr286) (1:500, #PA1-4614), IP3 receptors (1:500, #PA5-96855) and phospho-IP3R (Ser1756) (1:1000, # PA5-101050).

• From Cell Signaling Technology, Danvers, MA, USA: AMPK (1:1000, #2532) and phospho-AMPK (Thr172) (1:500, #2535), CaMK II (1:1000, #3362), eEF2 (1:1000, #2332) and p-eEF2 (1:1000, #2331), GAPDH (1:5000, # 2118), S6 (1:1000, #2217) and pS6 (Ser240/244) (1:1000, #5364).

• From Cloud-clone, Wuhan, China: JC1 (1:1000, #PAC550Hu01).

• From Kerafast Inc., Boston, MA, USA: puromycin (1:3000, #EQ0001).

4.8. Muscle ATP Content Evaluation

ATP concentrations in frozen soleus muscle samples were quantified using a commercial Colorimetric/Fluorometric Assay Kit (MAK190; Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. This procedure followed the protocol previously established by Zaripova et al. [48].

4.9. Muscle Ca²⁺ Content Evaluation

Cytoplasmic and mitochondrial Ca²⁺ levels were assessed using Oregon Green BAPTA-1 (OGB-1) and Rhod-2 AM (Invitrogen, USA), respectively, following previously described methods [65]. Soleus muscle was attached to a Petri dish, washed with Ringer–Krebs solution (120 mM sodium chloride, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 25 mM sodium bicarbonate, and 5.5 mM D-glucose), and incubated for 40 min in the dark with a staining solution containing 5 μM Rhod-2-AM, 5 μM OGB-1, and 0.001% Pluronic. At room temperature, the positive charge of Rhod-2 AM facilitates its selective sequestration within the mitochondrial matrix [66].

The preliminary experiments showed that Rhod-2-AM fluorescence was co-localized with MitoTracker-stained (Thermo Fisher Scientific, Waltham, MA, USA) regions of skeletal muscle fibers. Stained muscles were washed for 30 min in Ringer–Krebs solution, placed under a confocal microscope LSM 900 (Zeiss, Oberkochen, Germany), and photographed for 10 min. Then, the muscle was incubated in Ca²⁺-free solution (10 mM K2EGTA, 100 mM KCl, and 10 mM MOPS, pH 7.2), supplemented with 10 μM ionomycin for 20 min. After this, the photographs were taken again without changing the instrument settings, as previously described [30]. The muscle was then incubated in a 40 μM Ca²⁺-containing solution (10 mM CaEGTA, 100 mM KCl, and 10 mM MOPS, pH 7.2), supplemented with 10 μM ionomycin for 20 min, and the photographs were taken again for the evaluation of OGB-1 maximal fluorescence levels. The levels of fluorescence of OGB-1 and Rhod-2 in zero Ca²⁺ and 40 μM Ca²⁺ solutions were used as loading controls for signal normalization. The levels of fluorescence were assessed using Zen 5 software (Zeiss, Oberkochen, Germany). The mean fluorescence intensity (IF) values were measured in at least 20 fibers for each sample. Carbogen was not administered to the muscles during incubation with calcium probes because, in vivo, both the oxygen supply and oxygen consumption of non-contracting muscle are very low. At room temperature, the PO₂ required for resting muscle metabolism is lower than the PO₂ in saline [67].

4.10. RNA Isolation and QRT-PCR

Total RNA isolation and reverse transcription (RT) were performed using the RNeasy Micro Kit (Qiagen, Hilden, Germany) as previously described [48]. RT was done using a RevertAid RT Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 0.5 micrograms of total RNA. Amplification was performed using Quantitect SYBR Green Master Mix (Syntol, Moscow, Russia) and 10 pM of each forward and reverse primer, as previously described [48]. Primers were synthesized by Syntol (Moscow, Russia). While both RPL19 and GAPDH were initially evaluated as internal controls with comparable results, RPL19 was selected as the reference gene for all subsequent normalization.

The primer sequences were as follows:

• MAFbx (sense: 5′-CTACGATGTTGCAGCCAAGA-3′/anti-sense: 5′-GGCAGTCGAGAAGTCCAGTC-3′).

• MuRF1 (sense: 5′-GCCAATTTGGTGCTTTTTGT-3′/anti-sense: 5′-AAATTCAGTCCTCTCCCCGT-3′).

• PGC1alpha (sense: 5′-ATGTGTCGCCTTCTTGCTCT-3′/anti-sense: 5′-ATCTACTGCCTGGGGACCTT-3′).

• RPL19 (sense: 5′-GTACCCTTCCTCTTCCCTATGC-3′/anti-sense: 5′-CAATGCCAACTCTCGTCAACAG-3′).

• Ubiquitin (sense: 5′-CACCAAGAAGGTCAAACAGGA-3′/anti-sense: 5′-GCAAGAACTTTATTCAAAGTGCAA-3′).

4.11. Statistical Evaluation

Statistical analyses were performed using GraphPad Prism software 10.6.0. In the present study, Western blot, immunostaining, and PCR data are presented as mean values ± standard error of the mean (SEM), while the data on body weights, muscle weights, lengths, indexes, and force data are shown as mean values ± standard deviation (SD). Group comparisons were conducted using one-way ANOVA followed by Tukey’s post hoc test. The Shapiro–Wilk test was applied to assess data normality. If normality was not met, nonparametric statistical methods were employed, specifically Kruskal–Wallis ANOVA followed by Dunnett’s post hoc test. A p-value of less than 0.05 was considered indicative of statistical significance. All PCR and Western blot analyses were repeated three times. For most of the analyses, the number of samples is 8 in each group; in Western blots, some samples were indicated as outliers by the ROUT method, so the number of analyzed samples is less than 8 (indicated in the figure legends). In the immunohistochemistry analysis, the number of samples is less than 8 in each group because of the shortage of some samples that were fully used for previous analyses.

5. Conclusions

Inhibition of DHPRs with nifedipine during three days of soleus muscle unloading blocks unloading-induced increases in the accumulation of ATP and ROS, prevents PGC1alpha mRNA expression and JP1 proteolysis, attenuates the decrease in CSA and the slow type to fast type shift in fibers, and prevents the increase in the content of intramitochondrial and myoplasmic calcium, which led to the maintenance of the maximum force of a single contraction. Thus, DHPRs participated in energy and Ca²⁺ metabolism and affected the functional properties of muscles.

Author Contributions

Conceptualization: T.L.N. Data Curation: T.L.N. Formal Analysis: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z. and T.L.N. Funding Acquisition: T.L.N. Investigation: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z. and T.L.N. Methodology: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z. and T.L.N. Project Administration: T.L.N. Resources: T.L.N. Supervision: T.L.N. Visualization: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z., T.Y.K. and T.L.N. Writing—Original Draft Preparation: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z., T.Y.K., B.S.S. and T.L.N. Writing—Review and Editing: K.A.S., S.A.T., D.A.S., R.O.B., K.A.Z., T.Y.K., B.S.S. and T.L.N. All authors have read and agreed to the published version of the manuscript. All authors agreed to be accountable for all aspects of this study. All persons designated as authors qualified for authorship. All those who qualified for authorship are listed.

Institutional Review Board Statement

The Committee on Bioethics of the Russian Academy of Sciences reviewed and approved all animal experiments for this study (protocol 673; 2 December 2024).

Informed Consent Statement

Not applicable.

Data Availability Statement

Original data are available from the corresponding author upon request.

Conflicts of Interest

The authors declare no competing or financial interests.

Funding Statement

This work was supported by the Russian Science Foundation (RSF; project No. 24-15-00088, TLN). None of the other authors of this manuscript had grant support for this study.