Fig. 3. CaMKII activation is unrelated to the membrane hyperpolarization. (A) CaMKII activity was assessed by measuring the ability of myoblast extracts to phosphorylate a specific peptide substrate in presence of 32P-ATP. Total cell extracts containing phosphatase inhibitors were prepared from proliferating myoblast (GM) or proliferating myoblast with 1.8 mM Ca²⁺ (GM_1.8), and from myoblast induced to differentiate for 1 or 24 hours in differentiation medium (DM), or in DM containing 10 mM Cs⁺ (Cs), 116 mM KCl (KCl), 15 µM mM Ca²⁺ (DM_low) or 0.7 mM Ca²⁺ (DM_0.7). Endogenous CaMKII activity (activity in cultured) was assessed in absence of added Ca²⁺ or calmodulin; total CaMKII activity (maximum activity of the myoblast sample) was assessed after addition of 5 mM Ca²⁺ and 5 µM calmodulin. Specific activation of CaMKII was calculated as the ratio between the endogenous and the total activity. For each experiment, the ratio obtained with myoblasts maintained in differentiation medium containing 15 µM BAPTA-AM was set to 1. Results are expressed as mean±s.e.m. (B) CaMKII activation in high Ca²⁺ proliferating medium does not induce myoblast differentiation. Differentiation is expressed as a percentage of myogenin- and MEF2-positive nuclei. Results are expressed as mean±s.e.m.