Ca2+ transient‐dependent changes in embryonic myocyte proteins during myofibrillogenesis: a proteomic analysis

Abstract

Spontaneous intracellular calcium (Ca2+) signals, called transients, play a critical role during skeletal muscle myofibrillogenesis. In embryonic skeletal muscle, several classes of Ca2+ transients occur both in vivo and in culture, and blocking their production prevents de novo sarcomere assembly. Since Ca2+ signaling is paramount to a variety of cell signaling events, it is expected that perturbing these events would effect protein expression and/or posttranslational modification. We implemented a proteomics screen to assess global protein differences. We are using two-dimensional (2D) electrophoresis to study both the absolute protein amount as well as any modification that would affect pI. Protein lysates from whole embryos were optimized and enriched for myofibrillar proteins. Several protein spots showing a presumptive increase in Ca2+ dependent phosphorylation were identified by MS/MS. Other MS identified proteins showed marked decrease in sarcomeric incorporation. For example, the fast skeletal regulatory light chain (MLC2f) showed a decrease in both sarcomeric incorporation and phosphorylation when Ca2+ transients were inhibited. In order to assess whether MLC2f expression and/or phosphorylation are necessary for myofibrillogenesis, MLC2f expression was knocked down using morpholino technology. Embryos were completely paralyzed but otherwise appeared normal. MLC2f knockdown resulted in complete inhibition of sarcomeric myosin organization. We are currently creating MLC2f constructs for rescue and sufficiency experiments using both phosphodominant and phosphonegative site-directed mutagenesis.