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. 2023 Sep 6;13(9):1354.
doi: 10.3390/biom13091354.

The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading

Kristina A Sharlo  1 , Irina D Lvova  1 , Sergey A Tyganov  1 , Ksenia A Zaripova  1 , Svetlana P Belova  1 , Tatiana Y Kostrominova  2 , Boris S Shenkman  1 , Tatiana L Nemirovskaya  1
Affiliations

The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading

Kristina A Sharlo et al. Biomolecules. .

Abstract

Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.

Keywords: AMPK; ATP; MAFbx; MuRF1; muscle atrophy; soleus muscle unloading.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Soleus muscle weight normalized to rat total body weight (A) and soleus muscle fatigue index (B) in control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05.
Figure 2
Figure 2
Evaluation of CaMKII phosphorylation (A), CaN A (B), calpain-1 (C), SERCA1 (D), and SERCA2 (E). mRNA expression, and oxidized tropomyosin content (F) in soleus muscle of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). The content of phosphorylated CaMKII was normalized to the level of total CaMKII in each sample (A). The content of oxidized tropomyosin was normalized to the level of reduced tropomyosin in each sample (D). Levels of SERCA1 (B), SERCA2 (C), CaN (E), and calpain-1 (F) mRNA were normalized to the RPL19 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05. Original Western blot images are in the Supplementary File S1.
Figure 3
Figure 3
Evaluation of the relative content of fast (A) and slow (B) muscle fibers and CSA of fast (C) and slow (D) muscle fibers and a representative picture of fast and slow myosin immunostaining in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05.
Figure 4
Figure 4
mRNA expression of MyHC Iβ (A), MyHC IIa (B), MyHC IId/x (C), and MyHC IIb (D). Myosin in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). Levels of MyHC isoforms mRNA were normalized to the RLP19 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, p < 0.05.
Figure 5
Figure 5
Evaluation of the relative content of NFATc1 using immunostaining (A), and the level of MCIP1.4 mRNA expression (B) in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). Levels of MCIP1.4 mRNA (B) were normalized to RPL19 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05.
Figure 6
Figure 6
Evaluation of the relative level of JNK1/2 (A) and p38 (B) phosphorylation in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). The content of p-JNK1/2 (Thr 183, Tyr 185) and p-p38 were normalized to the levels of total JNK ½ and total p38 in each sample, respectively. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05. Original Western blot images are in the Supplementary File S1.
Figure 7
Figure 7
Evaluation of mRNA expression of PGC1a (A), COXI (B), COXII (C), and COXIV (D) in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). mRNA expression levels of PGC1α and COX isoforms were normalized to the RPL13 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05.
Figure 8
Figure 8
Evaluation of mtDNA content (A), TOM20 protein content (B), and mitofusin-1 (C) and mitofusin-2 (D) mRNA expression in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). TOM20 protein expression was normalized to the GAPDH expression in each sample. mRNA expression levels of mitofusins were normalized to the RPL19 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05. Original Western blot images are in the Supplementary File S1.
Figure 9
Figure 9
Evaluation of MuRF-1 (A) and MAFbx (B) mRNA expression in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). mRNA expression levels of MuRF-1 and MAFbx were normalized to the RPL19 mRNA expression in each sample. N = 8. * indicates a significant difference from the control, p < 0.05.
Figure 10
Figure 10
Evaluation of p70S6k (A) and eEF2 (B) phosphorylation levels in soleus muscles of control rats (C), rats with 7 days of unloading (7HS), and 7 days of HS with CDN1163 (7HSC). The phosphorylation levels of p70S6k and eEF2 were normalized to the levels of non-phosphorylated p70S6k and eEF2 proteins, respectively, in each sample. N = 8. * indicates a significant difference from the control, ** indicates a significant difference from the HS group, p < 0.05. Original Western blot images are in the Supplementary File S1.
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