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

• Published in: Free radical biology & medicine Vol. 224; pp. 182 - 189
• Main Authors: Martinez-Canton, Miriam, Gallego-Selles, Angel, Galvan-Alvarez, Victor, Garcia-Gonzalez, Eduardo, Garcia-Perez, Giovanni, Santana, Alfredo, Martin-Rincon, Marcos, Calbet, Jose A.L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2024
• Subjects: Calcium/calmodulin-dependent protein kinase II isoforms; Exercise; Human; Immunoblotting; Signalling; Skeletal muscle; Human; HDAC4; NF-κΒ; RT; Calcium/calmodulin-dependent protein kinase II isoforms; Immunoblotting; MW; Signalling; Skeletal muscle; MHC-IIx; TBS-T; Exercise; CaMKII; NRF2; VL; ROS; Keap1
• ISSN: 0891-5849; 1873-4596; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2024.08.030

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, δD, δA, γD, γB and βM 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 βM CaMKII and allows distinguishing between γ/δ and δD CaMKII isoforms. The corresponding molecular weight for the CaMKII isoforms resolved were: δD, at 54.2 ± 2.1 kDa; γ/δ, at 59.0 ± 1.2 kDa and 61.6 ± 1.3 kDa; and βM isoform, at 76.0 ± 1.8 kDa. Some tested antibodies showed high specificity for the δD, 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. [Display omitted] •CaMKII, an enzyme coded by four genes (α, β, γ, δ), is activated by ROS and Ca2+ transients during exercise.•Using eight commercial antibodies, we describe a protocol to resolve CaMKII isoforms in human skeletal muscle by Western blot.•The observed molecular weights of CaMKII isoforms were βM: 76±1.8, γ/δ: 59±1.2/61.6±1.3, and δD: 54.2±2.1 kDa, respectively.•Commercial antibodies with high specificity for the δD CaMKII isoform have been identified.•We show how exercise-induced posttranslational modifications can be used to validate CaMKII antibody specificity.