Regulation of Ca2+ handling by phosphorylation status in mouse fast- and slow-twitch skeletal muscle fibers

Abstract

The effects of phosphorylation status on Ca2+release and Ca2+ removal were studied in fast-twitch flexor digitorum brevis and slow-twitch soleus skeletal muscle fibers enzymatically isolated from wild-type and phospholamban knockout (PLBko) mice. In all fibers the adenosine 3′,5′-cyclic monophosphate-dependent protein kinase (PKA) inhibitor H-89 decreased the peak amplitude of the intracellular Ca2+ concentration ([Ca2+]) transient for a single action potential, and the PKA activator dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP) reversed this effect, indicating modulation of Ca2+release by phosphorylation status in all fibers. H-89 decreased the decay rate constant of the [Ca2+] transient and DBcAMP reversed this effect only in phospholamban-expressing fibers (wild-type soleus), indicating modulation of Ca2+ removal only in the presence of phospholamban. A high basal level of PKA phosphorylation in soleus fibers maintained under our control conditions was indicated by the lack of effect of direct application of DBcAMP on Ca2+ release or Ca2+ removal in wild-type or PLBko soleus fibers and was confirmed by analysis of phospholamban from wild-type soleus fibers.