Exploration of Creatine Kinase Proteoforms in C2C12 Myogenesis

In skeletal muscle, creatine (Cr) is phosphorylated and then serves as a high-energy phosphate store. Creatine kinases (CKs) catalyze the reversible transfer of phosphate from phosphocreatine (PCr) to ADP to make ATP, which can be used as a source of chemical energy for muscle contraction. There are three types of CKs in skeletal muscle: brain CK (CK-B), a cytoplasmic form of CK found in brain, heart and skeletal muscle; sarcomeric mitochondrial CK (CK-mt2), found in cardiac and skeletal muscle; and muscle-specific CK (CK-M), a cytosolic for of CK found only in skeletal muscle. This study investigated the abundance and phosphorylation of the three CKs throughout skeletal muscle myogenesis using C2C12 murine myoblasts as a model. C2C12 cells were harvested at the myoblast (D0), early myotube (D5), and late myotube (D9) stages and then analyzed. 1D gel electrophoresis, followed by Western blotting, was used to calculate the relative abundance of each CK between the three stages of myogenesis, and 2D gel electrophoresis, followed by Western blotting, was used to visualize the number of phosphorylation events on each of the creatine kinases in the three stages of C2C12 myogenesis. The relative abundance of CK-B was found to increase between D0 and D5 but remain the same thereafter; there was no significant change to the abundance of CK-mt2; and CK-M expression increased between every stage of myogenesis. As for phosphorylation, there were fewer concurrent phosphorylations on CK-B at D5 than at D0; there were consistently six phosphorylation states of CK-mt2 throughout myogenesis; and the number of phosphorylations on CK-M decreased between D5 and D9. Further evidence about the specific sites of phosphorylation on each of the CKs is needed to draw conclusions about the effects of phosphorylation on those enzymes.