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 (α, β, γ, and δ). In rat skeletal muscle, δ_D, δ_A, γ_D, γ_B and β_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 β_M CaMKII and allows distinguishing between γ/δ and δ_D CaMKII isoforms. The corresponding molecular weight for the CaMKII isoforms resolved were: δ_D, at 54.2 ± 2.1 kDa; γ/δ, at 59.0 ± 1.2 kDa and 61.6 ± 1.3 kDa; and β_M isoform, at 76.0 ± 1.8 kDa. Some tested antibodies showed high specificity for the δ_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.