GPCR Stimulation Modulates Camkii Translocation and Targeting in Cardiomyocytes

Abstract

Calcium/Calmodulin-dependent protein kinase II (CaMKIIδ) is a key regulator of cardiac function via its influence on ion channels, calcium handling, contraction, and transcription. Our group recently showed that pacing-induced activation of CaMKIIδ in cardiomyocytes enhances its mobility and rapid translocation to its extra-dyadic targets, providing a mechanism for broad myocyte target phosphorylation. It is not known whether GPCR-induced CaMKIIδ activation likewise stimulates CaMKII mobility. Here we used immunocytochemistry and protein expansion microscopy of endogenous CaMKII and fluorescence recovery after photobleach (FRAP) of GFP-tagged CaMKIIδ isoforms to measure CaMKIIδ mobility and intracellular targeting in intact adult cardiomyocytes. At low [Ca] (BAPTA-treated cells) and at rest, CaMKII was concentrated at the Z-lines but spread throughout the sarcomere with isoproterenol (ISO, 1 µM) or Angiotensin II (Ang II,100 nM) stimulation for 10 min. FRAP experiments likewise showed increased mobility for δC-GFP (τ for BAPTA ∼ 35s, ISO ∼ 16s and AngII ∼13s) and δB-GFP (τ for respectively BAPTA ∼ 21s, ISO ∼ 10s and AngII ∼10s). These effects were more pronounced upon chronic (20-24 hr) exposure to 0.5 Hz pacing with or without AngII or ISO. FRAP was significantly faster with pacing (τ ∼ 12s vs. rest ∼ 17s) and even more so with ISO or AngII exposure for δC-GFP (τ ISO ∼ 10s and AngII ∼10s) and δB-GFP (ISO ∼ 7s). The contribution of specific activating posttranslational modifications were also assessed using PTM-resistant CaMKIIδ constructs. In conclusion, acute and chronic activation of CaMKII via GPCR stimulation enhances its translocation which could explain CaMKII's regulation of pathological conditions, and widespread signaling within the myocyte.