<b>Abstract</b>— <p>Early changes in the myosin genes expression pattern lead to slow-to-fast fiber type shift, functional and metabolic alterations in skeletal muscles. A detailed understanding of myosin isoform transcriptional regulation could be the key for effective countermeasures against the unloading-induced negative changes. Slow-type myosin mRNA expression changes non-linearly in postural soleus muscle during rat hindlimb suspension, with a partial re-activation of slow-type myosin expression between the 2nd and the 4th&#xa0;day. Calcium ions accumulation is a well-studied activator of slow-type myosin expression, and after the second day of rat hindlimb suspension calcium was shown to increase in soleus muscle myoplasm. We suggested that calcium ions accumulation could contribute to the transient re-activation in slow-type myosin gene expression after the 2nd day of rat hindlimb suspension via activation of calcineurin/NFATc1 and/or blocking of HDAC4/MRF4 signaling pathways. If this hypothesis is correct, the prevention of the unloading-induced calcium accumulation by L-type channels inhibitor nifedipine would be expected to downregulate the expression of slow-type myosin. To test our hypothesis, the experiment with 3-day rat hindlimb suspension (HS) accompanied by nifedipine (L-type calcium channels blocker) administration was conducted. After 3 days of HS, we observed an increase in slow-type myosin mRNA expression and an increase in NFATc1 MCIP1.4 reporter activity, which was not observed in the group receiving nifedipine. In the group with 3-day rat hindlimb suspension and receiving nifedipine (group HS + Nifedipine), we also observed an accumulation of HDAC4 and MRF4 in the nucleus and a decrease in NFATc1 content in the nucleus compared to 3 days of HS. These data indicate that calcium ions accumulation after 3 days of HS contributes to the reactivation of slow myosin expression and activates both HDAC4-dependent and NFATc1-dependent signaling pathways during unloading.</p>