Expression of Sarco(endo)Plasmic Reticulum Ca²⁺‐Atpase Slow (SERCA2) Isoform in Regenerating Rat Soleus Skeletal Muscle Depends on Nerve Impulses

Abstract

We have examined the influence of innervation on the expression of different isoforms of sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) in regenerating rat slow twitch muscle. The process of degeneration/ regeneration was induced by injection of bupivacaine into rat soleus muscle under four different conditions: (1) in the presence of intact motor nerves, (2) after surgical denervation, (3) with nerve impulse conduction blocked by the Na+‐channel blocker tetrodotoxin (TTX), and (4) with the axoplasmic flow blocked by vinblastine. Expression of SERCA isoforms was visualized by immunohistochemical and Western blot analysis. In regenerating innervated muscle, SERCA1, the isoform normally expressed in fast twitch fibres, was present after 5 days and was then progressively replaced by SERCA2, the isoform typical of slow twitch fibres. The maximum Ca2+ uptake, measured in single skinned fibres regenerating for 10‐21 days, was similar to that of slow adult fibres and significantly lower than that of fast adult fibres. Denervation or TTX treatment prevented the expression of the SERCA2 isoform. Conversely, vinblastine did not affect the expression of SERCA isoforms. These data indicate that nerve impulses play a determinant role in the expression of the SERCA2 isoform.

Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

“…3). In contrast, SERCA2a, an isoform only present in rat type I fibers [42,43], was not detected in either four equivalents of skinned fibers or SL cuffs despite positive detection in 7.5 lg of soleus protein (Fig. 3).…”

Sphingosine 1-phosphate signaling is involved in skeletal muscle regeneration

“…Muscle tissue injury and acute degeneration was induced bilaterally in the soleus muscles by injecting, through a small cutaneous incision, 0.2 ml of 0.5% bupivacaine solution (Marcaina, Astra), as previously published (13,28,30). In the contralateral soleus, together with the myotoxic drug, 50 M S1P, 50 M Sph, or 20 M sphingosylphosphorylcholine (SPC) was also injected.…”

“…More than 300 fibers per muscle were evaluated. Immunofluorescence staining was performed as previously described (13,47). Briefly, muscle sections were incubated with selected antibodies under the conditions described above.…”

Expression of SERCA2a is not regulated by calcineurin or upon mechanical unloading in skeletal muscle regeneration

“…The slow type of myosin heavy chain isoform (MyHCI) and the slow type of sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) are characteristic markers of slow‐twitch skeletal muscle and are normally co‐expressed in slow type fibers. Further, MyHCI and SERCA2a levels mostly change in parallel during fast to slow transition of fiber types [1], upon denervation [2], during regeneration [3–5], and in adaptations to overload [6] and passive stretch [7]. The expression of the slow type MyHCI is entirely dependent on the corresponding slow type of innervation and is mainly regulated at the gene transcriptional level [8,9].…”