CaMKII protein expression and phosphorylation in human skeletal muscle by immunoblotting: Isoform specificity

Abstract

Calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) is activated during exercise by reactive oxygen species (ROS) and Ca2+ transients initiating muscle contraction. CaMKII modulates antioxidant, inflammatory, metabolic and autophagy signalling pathways. CaMKII is coded by four homologous genes (alpha, beta, gamma, and delta). In rat skeletal muscle, delta D, delta A, gamma D, gamma B and beta M have been described while different characterisations of human skeletal muscle CaMKII isoforms have been documented. Precisely discerning between the various isoforms is pivotal for understanding their distinctive functions and regulatory mechanisms in response to exercise and other stimuli. This study aimed to optimize the detection of the different CaMKII isoforms by western blotting using eight different CaMKII commercial antibodies in human skeletal muscle. Exercise-induced posttranslational modifications, i.e. phosphorylation and oxidations, allowed the identification of specific bands by multitargeting them with different antibodies after stripping and reprobing. The methodology proposed has confirmed the molecular weight of beta M CaMKII and allows distinguishing between gamma/delta and delta D CaMKII isoforms. The corresponding molecular weight for the CaMKII isoforms resolved were: delta D, at 54.2 f 2.1 kDa; gamma/delta, at 59.0 f 1.2 kDa and 61.6 f 1.3 kDa; and beta M isoform, at 76.0 f 1.8 kDa. Some tested antibodies showed high specificity for the delta D, the most responsive isoform to ROS and intracellular Ca2+ transients in human skeletal muscle, while others, despite the commercial claims, failed to show such specificity.